Methods and Compositions for the Production of Orthogonal tRNA-Aminoacyl tRNA Synthetase Pairs

ABSTRACT

This invention provides compositions and methods for generating components of protein biosynthetic machinery including orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases. Methods for identifying orthogonal pairs are also provided. These components can be used to incorporate unnatural amino acids into proteins in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 11/978,154, filed Oct.26, 2007, which is a continuation of Ser. No. 11/254,170, filed Oct. 18,2005, which is a continuation of U.S. patent application Ser. No.10/126,931 filed Apr. 19, 2002 which claims priority to U.S. provisionalpatent application Ser. No. 60/285,030, filed Apr. 19, 2001, and U.S.patent application Ser. No. 60/355,514, filed Feb. 6, 2002, thespecifications of all of which are incorporated herein in theirentirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

The invention was made with United States Government support under GrantNo. N0001498F0402 from the Office of Naval Research, Contract No. NIHGM62159 from the National Institutes of Health, and Contract Nos.DE-FG03-00ER45812, DE-AC03-76SF00098 from the Department of Energy. TheUnited States Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to the field of translation biochemistry. Inparticular, the invention relates to methods for producing mutatedorthogonal tRNAs, mutated orthogonal aminoacyl-tRNA synthetases, andpairs thereof. The invention also provides methods for identifyingorthogonal pairs, which are used for the incorporation of unnaturalamino acids into proteins in vivo, and related compositions.

BACKGROUND OF THE INVENTION

Proteins carry out virtually all of the complex processes of life, fromphotosynthesis to signal transduction and the immune response. Tounderstand and control these intricate activities, a betterunderstanding of the relationship between the structure and function ofproteins is needed.

Unlike small organic molecule synthesis wherein almost any structuralchange can be made to influence functional properties of a compound, thesynthesis of proteins is limited to changes encoded by the twentynatural amino acids. The genetic code of every known organism, frombacteria to human, encodes the same twenty common amino acids. Theseamino acids can be modified by post-translational modification ofproteins, e.g., glycosylation, phosphorylation or oxidation, or in rarerinstances, by the enzymatic modification of aminoacylated suppressortRNAs, e.g., in the case of selenocysteine. Nonetheless, polypeptides,which are synthesized from only these 20 simple building blocks, carryout all of the complex processes of life.

Both site-directed and random mutagenesis, in which specific amino acidsin a protein can be replaced with any of the other nineteen common aminoacids, have become important tools for understanding the relationshipbetween the structure and function of proteins. These methodologies havemade possible the generation of proteins with enhanced properties,including stability, catalytic activity and binding specificity.Nevertheless, changes in proteins are limited to the 20 common aminoacids, most of which have simple functional groups. See Knowles, J. R.Tinkering with enzymes: what are we learning? Science, 236:1252-1258(1987); and, Zoller, M. J., Smith, M. Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods Enzymol,100:468-500 (1983). By expanding the genetic code to include additionalamino acids with novel biological, chemical or physical properties, theproperties of proteins, e.g., the size, acidity, nucleophilicity,hydrogen-bonding, hydrophobic properties, etc., can be modified ascompared to a protein composed of only amino acids from the 20 commonamino acids, e.g., as in a naturally occurring protein.

Several strategies have been employed to introduce unnatural amino acidsinto proteins. The first experiments involved the derivatization ofamino acids with reactive side-chains such as Lys, Cys and Tyr, forexample, the conversion of lysine to N^(s)-acetyl-lysine. Chemicalsynthesis also provides a straightforward method to incorporateunnatural amino acids, but routine solid-phase peptide synthesis isgenerally limited to small peptides or proteins with less than 100residues. With the recent development of enzymatic ligation and nativechemical ligation of peptide fragments, it is possible to make largerproteins, but such methods are not easily scaled. See, e.g., P. E.Dawson and S. B. H. Kent, Annu. Rev. Biochem., 69:923 (2000). A generalin vitro biosynthetic method in which a suppressor tRNA chemicallyacylated with the desired unnatural amino acid is added to an in vitroextract capable of supporting protein biosynthesis, has been used tosite-specifically incorporate over 100 unnatural amino acids into avariety of proteins of virtually any size. See, e.g., V. W. Cornish, D.Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995, 34:621(1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244 182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111 8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction. Although these studies demonstrate that the proteinbiosynthetic machinery tolerates a wide variety of amino acid sidechains, the method is technically demanding, and yields of mutantproteins are low.

Over 50 years ago, it was found that many analogs of natural amino acidsinhibit the growth of bacteria. Analysis of the proteins produced in thepresence of these amino acid analogs revealed that they had beensubstituted for their natural counterparts to various extents. See,e.g., M. H. Richmond, Bacteriol. Rev., 26:398 (1962). This occursbecause the aminoacyl-tRNA synthetase, the enzyme responsible for theattachment of the correct amino acid to its cognate tRNA, cannotrigorously distinguish the analog from the corresponding natural aminoacid. For instance, norleucine is charged by methionyl-tRNA synthetase,and p-fluorophenylalanine is charged by phenylalanine-tRNA synthetase.See, D. B. Cowie, G. N. Cohen, E. T. Bolton and H. deRobichon-Szulmajster, Biochim. Biophys. Acta, 1959, 34:39 (1959); and,R. Munier and G. N. Cohen, Biochim. Biophys. Acta, 1959, 31:378 (1959).

An in vivo method, termed selective pressure incorporation, was laterdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage λ lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been inserted in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also been insertedefficiently, allowing for additional modification of proteins bychemical means. See, e.g., J. C. M. van Hest and D. A. Tirrell, FEBSLett., 428:68 (1998); J. C. M. van Hest, K. L. Kiick and D. A. Tirrell,J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A. Tirrell,Tetrahedron, 56:9487 (2000).

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,requires high selectivity to insure the fidelity of protein translation.Therefore, the range of chemical functionality accessible via this routeis limited. For instance, although thiaproline can be incorporatedquantitatively into proteins, oxaproline and selenoproline cannot. See,N. Budisa, C. Minks, F. J. Medrano, J. Lutz, R. Huber and L. Moroder,Proc. Natl. Acad. Sci. USA, 95:455 (1998). One way to expand the scopeof this method is to relax the substrate specificity of aminoacyl-tRNAsynthetases, which has been achieved in a limited number of cases. Forexample, it was found that replacement of Ala²⁹⁴ by Gly in Escherichiacoli phenylalanyl-tRNA synthetase (PheRS) increases the size ofsubstrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS, Nishimura, J. Biol. Chem., 275:40324 (2000).

The fidelity of aminoacylation is maintained both at the level ofsubstrate discrimination and proofreading of non-cognate intermediatesand products. Therefore, an alternative strategy to incorporateunnatural amino acids into proteins in vivo is to modify synthetasesthat have proofreading mechanisms. These synthetases cannot discriminateand therefore activate amino acids that are structurally similar to thecognate natural amino acids. This error is corrected at a separate site,which deacylates the mischarged amino acid from the tRNA to maintain thefidelity of protein translation. If the proofreading activity of thesynthetase is disabled, structural analogs that are misactivated mayescape the editing function and be incorporated. This approach has beendemonstrated recently with the valyl-tRNA synthetase (ValRS). See, V.Doring, H. D. Mootz, L. A. Nangle, T. L. Hendrickson, V. deCrecy-Lagard, P. Schimmel and P. Marliere, Science, 292:501 (2001).ValRS can misaminoacylate tRNAVal with Cys, Thr, or aminobutyrate (Abu);these noncognate amino acids are subsequently hydrolyzed by the editingdomain. After random mutagenesis of the Escherichia coli chromosome, amutant Escherichia coli strain was selected that has a mutation in theediting site of ValRS. This edit-defective ValRS incorrectly chargestRNAVal with Cys. Because Abu sterically resembles Cys (—SH group of Cysis replaced with —CH₃ in Abu), the mutant ValRS also incorporates Abuinto proteins when this mutant Escherichia coli strain is grown in thepresence of Abu. Mass spectrometric analysis shows that about 24% ofvalines are replaced by Abu at each valine position in the nativeprotein.

At least one major limitation of the methods described above is that allsites corresponding to a particular natural amino acid throughout theprotein are replaced. The extent of incorporation of the natural andunnatural amino acid may also vary—only in rare cases can quantitativesubstitution be achieved since it is difficult to completely deplete thecognate natural amino acid inside the cell. Another limitation is thatthese strategies make it difficult to study the mutant protein in livingcells, because the multi-site incorporation of analogs often results intoxicity. Finally, this method is applicable in general only to closestructural analogs of the common amino acids, again becausesubstitutions must be tolerated at all sites in the genome.

Solid-phase synthesis and semi-synthetic methods have also allowed forthe synthesis of a number of small proteins containing novel aminoacids. For example, see the following publications and references citedwithin: Crick, F. J. C., Barrett, L. Brenner, S. Watts-Tobin, R. Generalnature of the genetic code for proteins. Nature, 192:1227-1232 (1961);Hofmann, K., Bohn, H. Studies on polypeptides. XXXVI. The effect ofpyrazole-imidazole replacements on the S-protein activating potency ofan S-peptide fragment, J. Am. Chem., 5914-5919 (1966); Kaiser, E. T.Synthetic approaches to biologically active peptides and proteinsincluding enzymes, Acc. Chem. Res., 47-54 (1989); Nakatsuka, T., Sasaki,T., Kaiser, E. T. Peptide segment coupling catalyzed by thesemisynthetic enzyme thiosubtilisin, J. Am. Chem. Soc., 109:3808-3810(1987); Schnolzer, M., Kent, S B H. Constructing proteins by dovetailingunprotected synthetic peptides: backbone-engineered HIV protease,Science, 256(5054):221-225 (1992); Chaiken, I. M. Semisynthetic peptidesand proteins, CRC Crit. Rev. Biochem., 11(3):255-301 (1981); Offord, R.E. Protein engineering by chemical means? Protein Eng., 1(3):151-157(1987); and, Jackson, D. Y., Burnier, J., Quan, C., Stanley, M., Tom,J., Wells, J. A. A Designed Peptide Ligase for Total Synthesis ofRibonuclease A with Unnatural Catalytic Residues, Science,266(5183):243-247 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,283(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Rev. Biochem.,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenzyme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J. Biol. Chem.,243(24):6392-6401 (1968); Polgar, L. B., M. L. A new enzyme containing asynthetically formed active site. Thiol-subtilisin. J. Am. Chem. Soc.,88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G. Schultz, P. G.Introduction of nucleophiles and spectroscopic probes into antibodycombining sites, Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev. Biochem., 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotrophic for a particular aminoacid. See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz,P. G. A general method for site-specific incorporation of unnaturalamino acids into proteins, Science, 244:182-188 (1989); M. W. Nowak, etal., Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem. Soc.,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., 301-336 (1992); and,Mendel, D., Cornish, V. W. & Schultz, P. G. Site-Directed Mutagenesiswith an Expanded Genetic Code, Annu. Rev. Biophys. Biomol. Struct. 24,435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.

Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′, 3′ Exonuclease inphosphorothioate-based oligonucleotide-directed mutagenesis, NucleicAcids Res., 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³1-1]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,197-200 (1992).

In general, these in vitro approaches are limited by difficulties inachieving site-specific incorporation of the amino acids, by therequirement that the amino acids be simple derivatives of the commontwenty amino acids or problems inherent in the synthesis of largeproteins or peptide fragments.

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserted the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of α-hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms, see, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

However, there are limitations microinjection method, e.g., thesuppressor tRNA has to be chemically aminoacylated with the unnaturalamino acid in vitro, and the acylated tRNA is consumed as astoichiometric reagent during translation and cannot be regenerated.This limitation results in poor suppression efficiency and low proteinyields, necessitating highly sensitive techniques to assay the mutantprotein, such as electrophysiological measurements. Moreover, thismethod is only applicable to cells that can be microinjected.

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers the advantages of high yields of mutant proteins,technical ease, the potential to study the mutant proteins in cells orpossibly in living organisms and the use of these mutant proteins intherapeutic treatments. The ability to include unnatural amino acidswith various sizes, acidities, nucleophilicities, hydrophobicities, andother properties into proteins can greatly expand our ability torationally and systematically manipulate the structures of proteins,both to probe protein function and create new proteins or organisms withnovel properties. However, the process is difficult, because the complexnature of tRNA-synthetase interactions that are required to achieve ahigh degree of fidelity in protein translation. Therefore, improvementsto the process are needed to provide more efficient and effectivemethods to alter the biosynthetic machinery of the cell. The presentinvention addresses these and other needs, as will be apparent uponreview of the following disclosure.

SUMMARY OF THE INVENTION

The present invention provides compositions of components used inprotein biosynthetic machinery, which include orthogonaltRNA-aminoacyl-tRNA synthetase pairs and the individual components ofthe pairs. Methods for generating and selecting orthogonal tRNAs,orthogonal aminoacyl-tRNA synthetases, and pairs thereof that can use anunnatural amino acid are also provided. Compositions of the inventioninclude novel orthogonal tRNA-aminoacyl-tRNA synthetase pairs, e.g.,mutRNATyr-mutTyrRS pairs, mutRNALeu-mutLeuRS pairs, mutRNAThr-mutThrRSpairs, mutRNAGlu-mutGluRS pairs, and the like. The novel orthogonalpairs can be use to incorporate an unnatural amino acid in a polypeptidein vivo. Other embodiments of the invention include selecting orthogonalpairs.

Compositions of the present invention include an orthogonalaminoacyl-tRNA synthetase (O-RS), where the O-RS preferentiallyaminoacylates an orthogonal tRNA (O-tRNA) with an unnatural amino acid,optionally, in vivo. In one embodiment, the O-RS comprises a nucleicacid comprising a polynucleotide sequence selected from the groupconsisting of: SEQ ID NO: 4-34 (see, Table 5) and a complementarypolynucleotide sequence thereof. In another embodiment, the O-RS hasimproved or enhanced enzymatic properties, e.g., the K_(m) is higher orlower, the k_(cat) is higher or lower, the value of k_(cat)/K_(m) ishigher or lower or the like, for the unnatural amino acid compared to anaturally occurring amino acid, e.g., one of the 20 known amino acids.

The unnatural amino acids of the present invention encompass a varietyof substances. For example, they optionally include (but are not limitedto) such molecules as: an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcf3-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine. Additionally, other examples optionallyinclude (but are not limited to) an unnatural analogue of a tyrosineamino acid; an unnatural analogue of a glutamine amino acid; anunnatural analogue of a phenylalanine amino acid; an unnatural analogueof a serine amino acid; an unnatural analogue of a threonine amino acid;an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide,hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester,thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic,enone, imine, aldehyde, hydroxylamine, keto, or amino substituted aminoacid, or any combination thereof; an amino acid with a photoactivatablecross-linker; a spin-labeled amino acid; a fluorescent amino acid; anamino acid with a novel functional group; an amino acid that covalentlyor noncovalently interacts with another molecule; a metal binding aminoacid; a metal-containing amino acid; a radioactive amino acid; aphotocaged amino acid; a photoisomerizable amino acid; a biotin orbiotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; an amino acidcomprising polyethylene glycol; an amino acid comprising polyether; aheavy atom substituted amino acid; a chemically cleavable orphotocleavable amino acid; an amino acid with an elongated side chain;an amino acid containing a toxic group; a sugar substituted amino acid,e.g., a sugar substituted serine or the like; a carbon-linkedsugar-containing amino acid; a redox-active amino acid; an α-hydroxycontaining acid; an amino thio acid containing amino acid; anα,αdisubstituted amino acid; a β-amino acid; and a cyclic amino acidother than proline.

The present invention also includes a polypeptide comprising an aminoacid sequence encoded by a coding polynucleotide sequence which isselected from: a coding polynucleotide sequence selected from SEQ ID NO:4-34 (see, Table 5 for sequences); a coding polynucleotide sequenceencoding a polypeptide selected from SEQ ID NO: 35-66a polynucleotyidesequence which hybridizes under highly stringent conditions oversubstantially the entire length of such polynucleotide sequences; andcomplementary sequences of any of such sequences. Additionally, suchpolypeptide optionally encodes an orthogonal aminoacyl tRNA synthetaseand/or an amino acid sequence selected from SEQ ID NO:35 to SEQ IDNO:66.

The present invention also includes a nucleic acid comprising apolynucleotide sequence selected from the group consisting of: apolynucleotide sequence selected from SEQ ID NO:1 to SEQ ID NO:3 (or acomplementary polynucleotide sequence thereof) and a polynucleotidesequence which hybridizes under highly stringent conditions oversubstantially the entire length of such polynucleotide sequences. Suchnucleic acids also include wherein the polynucleotide sequence comprisesan orthogonal tRNA and/or wherein the polynucleotide sequence forms acomplementary pair with an orthogonal aminoacyl-tRNA synthetase (whichoptionally is selected from the those whose sequence is listed in SEQ IDNO:35 to SEQ ID NO:66.

Compositions of an orthogonal tRNA (O-tRNA) are also included, where theO-tRNA recognizes a selector codon and wherein the O-tRNA ispreferentially aminoacylated with an unnatural amino acid by anorthogonal aminoacyl-tRNA synthetase. In one embodiment, the O-tRNAcomprises a nucleic acid comprising a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO: 1-3 (see, Table 5) and acomplementary polynucleotide sequence thereof.

Selector codons of the present invention expand the genetic codonframework of protein biosynthetic machinery. For example, a selectorcodon includes, e.g., a unique three base codon (composed of natural orunnatural bases), a nonsense codon (such as a stop codon, e.g., an ambercodon, or an opal codon), an unnatural codon, a rare codon, a codoncomprising at least four bases, a codon comprising at least five bases,a codon comprising at least six bases, or the like.

In one embodiment, the O-tRNA (optionally comprising withincompositions) can include an orthogonal aminoacyl-tRNA synthetase(O-RS), e.g., where the O-tRNA and the O-RS are complementary, e.g., anO-tRNA/O-RS pair. In one embodiment, a pair comprises e.g., amutRNATyr-mutTyrRS pair, such as mutRNATyr-SS12TyrRS pair, amutRNALeu-mutLeuRS pair, a mutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRSpair, or the like. In another embodiment, the pair is other than amutRNAGln-mutGlnRS derived from Escherichia coli, a mutRNAAsp-mutAspRSderived from yeast or a mutRNAPheCUA-mutphenlalanineRS from yeast, wherethese pairs do not possess the properties of the pairs of the presentinvention.

The O-tRNA and the O-RS can be derived by mutation of a naturallyoccurring tRNA and RS from a variety of organisms. In one embodiment,the O-tRNA and O-RS are derived from at least one organism, where theorganism is a prokaryotic organism, e.g., Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, orthe like. Optionally, the organism is a eukaryotic organism, e.g.,plants (e.g., complex plants such as monocots, or dicots), algea, fungi(e.g., yeast, etc), animals (e.g., mammals, insects, arthropods, etc.),insects, protists, or the like. Optionally, the O-tRNA is derived bymutation of a naturally occurring tRNA from a first organism and theO-RS is derived by mutation of a naturally occurring RS from a secondorganism. In one embodiment, the O-tRNA and O-RS can be derived from amutated tRNA and mutated RS.

The O-tRNA and the O-RS also can optionally be isolated from a varietyof organisms. In one embodiment, the O-tRNA and O-RS are isolated fromat least one organism, where the organism is a prokaryotic organism,e.g., Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium, Escherichia coli, A. fulgidus, P. furiosus, P.horikoshii, A. pernix, T. thermophilus, or the like. Optionally, theorganism is a eukaryotic organism, e.g., plants (e.g., complex plantssuch as monocots, or dicots), algea, fungi (e.g., yeast, etc), animals(e.g., mammals, insects, arthropods, etc.), insects, protists, or thelike. Optionally, the O-tRNA is isolated from a naturally occurring tRNAfrom a first organism and the O-RS is isolated from a naturallyoccurring RS from a second organism. In one embodiment, the O-tRNA andO-RS can be isolated from one or more library (which optionallycomprises one or more O-tRNA and/or O-RS from one or more organism(including those comprising prokaryotes and/or eukaryotes).

In another aspect, the compositions of the present invention can be in acell. Optionally, the compositions of the present invention can be in anin vitro translation system.

Methods for generating components of the protein biosynthetic machinery,such as O-RSs, O-tRNAs, and orthogonal O-tRNA/O-RS pairs that can beused to incorporate an unnatural amino acid are provided in the presentinvention. Methods for selecting an orthogonal tRNA-tRNA synthetase pairfor use in in vivo translation system of an organism are also provided.The unnatural amino acids and selectors codons used in the methods aredescribed above and below.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O-RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, e.g., a prokaryotic organism, such asMethanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium, Escherichia coli, A. fulgidus, P. furiosus, P.horikoshii, A. pernix, T. thermophilus, or the like; (b) selecting(and/or screening) the library of RSs (optionally mutant RSs) formembers that aminoacylate an orthogonal tRNA (O-tRNA) in the presence ofan unnatural amino acid and a natural amino acid, thereby providing apool of active (optionally mutant) RSs; and/or, (c) selecting(optionally through negative selection) the pool for active RSs (e.g.,mutant RSs) that preferentially aminoacylate the O-tRNA in the absenceof the unnatural amino acid, thereby providing the at least onerecombinant O-RS; wherein the at least one recombinant O-RSpreferentially aminoacylates the O-tRNA with the unnatural amino acid.Recombinant O-RSs produced by the methods are also included in thepresent invention.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, e.g., alanine.

Libraries of mutant RSs can be generated using various mutagenesistechniques known in the art. For example, the mutant RSs can begenerated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, and by othermethods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, e.g., thataminoacylate an orthogonal tRNA (O-tRNA) in the presence of an unnaturalamino acid and a natural amino acid, includes: introducing a positiveselection or screening marker, e.g., an antibiotic resistance gene, orthe like, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, e.g., an amber, ochre, or opal codon;growing the plurality of cells in the presence of a selection agent;identifying cells that survive (or show a specific response) in thepresence of the selection and/or screening agent by suppressing the atleast one selector codon in the positive selection or screening marker,thereby providing a subset of positively selected cells that containsthe pool of active (optionally mutant) RSs. Optionally, the selectionand/or screening agent concentration can be varied.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is a∃-lactamase gene and the selector codon is an amber stop codon in the∃-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (e.g., a cell surface marker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the unnatural amino acid includes: introducing a negativeselection or screening marker with the pool of active (optionallymutant) RSs from the positive selection or screening into a plurality ofcells of a second organism, wherein the negative selection or screeningmarker comprises at least one selector codon (e.g., an antibioticresistance gene, e.g., a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a 1st media supplemented with the unnatural amino acid and ascreening or selection agent, but fail to survive or to show thespecific response in a 2nd media not supplemented with the unnaturalamino acid and the selection or screening agent, thereby providingsurviving cells or screened cells with the at least one recombinantO-RS. For example, a CAT identification protocol optionally acts as apositive selection and/or a negative screening in determination ofappropriate O-RS recombinants. For instance, a pool of clones isoptionally replicated on growth plates containing CAT (which comprisesat least one selctor codon) either with or without one or more unnaturalamino acid. Colonies growing exclusively on the plates containingunnatural amino acids are thus regarded as containing recombinant O-RS.In one aspect, the concentration of the selection (and/or screening)agent is varied. In some aspects the first and second organisms aredifferent. Thus, the first and/or second organism optionally comprises:a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungi, ayeast, an archaebacterium, a eubacterium, a plant, an insect, a protist,etc. In other embodiments, the screening marker comprises a fluorescentor luminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (e.g., negativelyselecting) the pool for active (optionally mutant) RSs includes:isolating the pool of active mutant RSs from the positive selection step(b); introducing a negative selection or screening marker, wherein thenegative selection or screening marker comprises at least one selectorcodon (e.g., a toxic marker gene, e.g., a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a 1st media not supplemented with the unnatural amino acid, but failto survive or show a specific screening response in a 2nd mediasupplemented with the unnatural amino acid, thereby providing survivingor screened cells with the at least one recombinant O-RS, wherein the atleast one recombinant O-RS is specific for the unnatural amino acid. Inone aspect, the at least one selector codon comprises about two or moreselector codons. Such embodiments optionally can include wherein the atleast one selector codon comprises two or more selector codons, andwherein the first and second organism are different (e.g., each organismis optionally, e.g., a prokaryote, a eukaryote, a mammal, an Escherichiacoli, a fungi, a yeast, an archaebacteria, a eubacteria, a plant, aninsect, a protist, etc.). Also, some aspects include wherein thenegative selction marker comprises a ribonuclease barnase gene (whichcomprises at least one selector codon). Other aspects include whereinthe screening marker optionally comprises a fluorescent or luminescentscreening marker or an affinity based screening marker. In theembodiments herein, the screenings and/or selections optionally includevariation of the screening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O-RS) can further comprise: (d)isolating the at least one recombinant O-RS; (e) generating a second setof O-RS (optionally mutated) derived from the at least one recombinantO-RS; and, (f) repeating steps (b) and (c) until a mutated O-RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, e.g., at least about twotimes. In one aspect, the second set of mutated O-RS derived from atleast one recombinant O-RS can be generated by mutagenesis, e.g., randommutagenesis, site-specific mutagenesis, recombination or a combinationthereof.

The stringency of the selection/screening steps, e.g., the positiveselection/screening step (b), the negative selection/screening step (c)or both the positive and negative selection/screening steps (b) and (c),in the above-described methods, optionally includes varying theselection/screening stringency. In another embodiment, the positiveselection/screening step (b), the negative selection/screening step (c)or both the positive and negative selection/screening steps (b) and (c)comprise using a reporter, wherein the reporter is detected byfluorescence-activated cell sorting (FACS) or wherein the reporter isdetected by luminescence. Optionally, the reporter is displayed on acell surface, on a phage display or the like and selected based uponaffinity or catalytic activity involving the unnatural amino acid or ananalogue. In one embodiment, the mutated synthetase is displayed on acell surface, on a phage display or the like.

The methods embodied herein optionally comprise wherein the unnaturalamino acid is selected from, e.g.: an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine. A recombinant O-RS produced by the methodsherein is also included in the current invention.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,e.g., a suppressor tRNA, from a first organism; (b) selecting (e.g.,negatively selecting) or screening the library for (optionally mutant)tRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from asecond organism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS (O-RS), thereby providingat least one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O-RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, e.g., prokaryotes (e.g.,Methanococcus jannaschii, Methanobacteium thermoautotrophicum,Escherichia coli, Halobacterium, etc.), eukaryotes, mammals, fungi,yeasts, archaebacteria, eubacteria, plants, insects, protists, etc.Additionally, the recombinant tRNA is optionally aminoacylated by anunnatrual amino acid, wherein the unnatural amino acid is biosynthesizedin vivo either naturally or through genetic manipulation. The unnaturalamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (e.g., negatively selecting) or screening thelibrary for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (step (b)) includes: introducing a toxicmarker gene, wherein the toxic marker gene comprises at least one of theselector codons (or a gene that leads to the production of a toxic orstatic agent or a gene esential to the organism wherein such marker genecomprises at least one selector codon) and the library of (optionallymutant) tRNAs into a plurality of cells from the second organism; and,selecting surviving cells, wherein the surviving cells contain the poolof (optionally mutant) tRNAs comprising at least one orthogonal tRNA ornonfunctional tRNA. For example, surviving cells can be selected byusing a comparison ratio cell density assay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O-RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (e.g., ∃-lactamase gene, comprising at least one of theselector codons, such as at least one amber stop codon) or a geneessential to the organism, or a gene that leads to detoxification of atoxic agent, along with the O-RS, and the pool of (optionally mutant)tRNAs into a plurality of cells from the second organism; and,identifying surviving or screened cells grown in the presence of aselection or screening agent, e.g., an antibiotic, thereby providing apool of cells possessing the at least one recombinant tRNA, where the atleast recombinant tRNA is aminoacylated by the O—RS and inserts an aminoacid into a translation product encoded by the positive marker gene, inresponse to the at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.Recombinant O-tRNAs produced by the methods of the present invention arealso included.

Methods for generating specific O-tRNA/O-RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutan) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS(O-RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant O-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the O-RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anunnatural amino acid and a natural amino acid, thereby providing a poolof active (optionally mutant) RSs; and, (f) negatively selecting orscreening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the unnatural amino acid, thereby providing the at least onespecific O-tRNA/O-RS pair, wherein the at least one specific O-tRNA/O-RSpair comprises at least one recombinant O-RS that is specific for theunnatural amino acid and the at least one recombinant O-tRNA. SpecificO-tRNA/O-RS pairs produced by the methods are included. For example, thespecific O-tRNA/O-RS pair can include, e.g., a mutRNATyr-mutTyrRS pair,such as a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and thrid organismare the same (e.g., Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, e.g., Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P.furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like.Alternatively, the organisms optionally comprise a eukaryotic organism,e.g., plants (e.g., complex plants such as monocots, or dicots), algae,protists, fungi (e.g., yeast, etc), animals (e.g., mammals, insects,arthropods, etc.), or the like. In another embodiment, the secondorganism is a prokaryotic organism, e.g., Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, e.g., a yeast, a animal cell, a plant cell, afungus, a mammalian cell, or the like. In various embodiments the firstand second organisms are different.

The various methods of the invention (above) optionally comprise whereinselecting or screening comprises one or more positive or negativeselection or screening, e.g., a change in amino acid permeability, achange in translation efficiency, and a change in translationalfidelity. Additionally, the one or more change is optionally based upona mutation in one or more gene in an organism in which an orthogonaltRNA-tRNA synthetase pair are used to produce such protein. Selectingand/or screening herein optionally comprises wherein at least 2 selectorcodons within one or more selection gene or within one or more screeninggene are use. Such multiple selector codons are optionally within thesame gene or within different screening/selection genes. Additionally,the optional multiple selector codons are optionally different selectorcodons or comprise the same type of selector codons.

Kits are an additional feature of the invention. For example, the kitscan include one or more translation system as noted above (e.g., acell), one or more unnatural amino acid, e.g., with appropriatepackaging material, containers for holding the components of the kit,instructional materials for practicing the methods herein and/or thelike. Similarly, products of the translation systems (e.g., proteinssuch as EPO analogues comprising unnatural amino acids) can be providedin kit form, e.g., with containers for holding the components of thekit, instructional materials forpracticing the methods herein and/or thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates site-specific incorporation ofunnatural amino acids into proteins in vivo. An orthogonalaminoacyl-tRNA synthetase aminoacylates an orthogonal tRNA with anunnatural amino acid. The acylated orthogonal tRNA inserts the unnaturalamino acid at the position specified by a selector codon, e.g., a uniquecodon, which is introduced into the gene encoding a protein of interest.

FIG. 2, Panel A and Panel B, schematically illustrates examples ofselection methods for active synthetases that aminoacylate withunnatural amino acids. Panel A illustrates the general selection/screenfor aminoacyl-tRNA synthetases with unnatural amino acids specificities.In the positive selection, active synthetases with either natural orunnatural amino acid specificities are identified; in the negativeselection, synthetases with natural amino acid specificities areeliminated. Only synthetases charging the orthogonal tRNA with theunnatural amino acid can survive both selections/screens. Panel Bschematically illustrates one embodiment of the selection/screen forsynthetases preferentially aminoacylating an O-tRNA with an unnaturalamino acid. For example, expression vectors containing an orthogonalsuppressor tRNA and a member of a library of mutated RS with a positiveselection marker, e.g., β-lactamase, with a selector codon, e.g., anamber codon, are introduced into an organism and grown in the presence aselector agent, e.g., ampicillin. The expression of the positiveselection marker allows the cell to survive in the selection agent.Survivors encode synthetases capable of charging any natural orunnatural amino acid (aa) onto the O-tRNA. The active synthetases aretransformed into a second strain in the expression vector, and anexpression vector with a negative selection marker, e.g., a toxic gene,such as barnase, that when expressed kills the cells, with one or moreselector codons, e.g., TAG. The cells are grown without the unnaturalamino acid. If the synthetase provided aminoacylates the O-tRNA with anatural amino acid, the negative selection marker is expressed and thecell dies. If the synthetase preferentially aminoacylates the O-tRNA, nonegative selection marker is expressed, because there is no unnaturalamino acid and the cell lives. This provides at least one orthogonalsynthetase that preferentially aminoacylates the O-tRNA with the desiredunnatural codon.

FIG. 3 illustrates site-specific mutations to generate directedlibraries for tyrosine analogues. Four regions of the protein areillustrated: residue 32, residues 67-70 (SEQ ID NO:80 for wild type,library 1, and library 2), residue 107, and residues 155-167 (SEQ IDNO:81 for wild type, SEQ ID NO:82 for library 1, SEQ ID NO:83 forlibrary 2, and SEQ ID NO:84 for library 3).

FIG. 4 illustrates a consensus sequence (SEQ ID NO:85 and SEQ ID NO:86)for pentafluorophenylalanine selection to generate directed librariesfor these analogues. Four regions of the protein are illustrated:residue 32, residues 67-70 (SEQ ID NO:80 for wild type, library 1, andlibrary 2), residue 107, and residues 155-167 (SEQ ID NO:81 for wildtype, SEQ ID NO:82 for library 1, SEQ ID NO:83 for library 2, and SEQ IDNO:84 for library 3).

FIG. 5 schematically illustrates the transplantation of one domain,e.g., the CPI domain, from one organism, e.g., Escherichia coli, to thesynthetase of other organism, e.g., Methanococcus jannaschii TyrRS.

FIG. 6 schematically illustrates the construction of chimericMethanococcus jannaschiil Escherichia coli synthetases. The E. coliTyrRS is SEQ ID NO:87, the H. sapiens TyrRS is SEQ ID NO:88, and the M.jannaschii TyrRS is SEQ ID NO:89.

FIG. 7 schematically illustrates the generation of a library of chimericsynthetases, e.g., Methanococcus jannaschii/Escherichia colisynthetases.

FIG. 8 schematically illustrates an example for selection of suppressortRNAs that are poor substrates for an endogenous synthetases, e.g., anEscherichia coli synthetase, and that are charged efficiently by acognate synthetase of interest. Expression vectors that contain a memberof a mutated tRNA library and another vector with a negative selectionmarker, e.g., a toxic gene, such as barnase, with one or more selectorcodons are introduced into a cell of an organism. Survivors of thenegative selection encode mutated tRNAs that are either orthogonal tothe organism or non-functional. The vectors from the survivors areisolated and transformed into other cells along with a positiveselection marker, e.g., β-lactamase gene, with a selector codon. Thecells are grown in the presence of a selection agent, e.g., ampicillin,and an RS from an organism from the same source, e.g., Methanococcusjannaschii, as the tRNA. Survivors of this selection encode mutant tRNAthat are orthogonal to the cell's synthetases, e.g., Escherichia coli'ssynthetases, and aminoacylated by RS from the same source as the tRNA.

FIG. 9, Panel A and B, schematically illustrates a mutatedanticodon-loop tRNA library, Panel A (SEQ ID NO:90), and a mutatedall-loop library, Panel B (SEQ ID NO:91), from Methanococcus jannaschiitRNA_(TyrCUA). Randomly mutated nucleotides (N) are shaded in black.

FIG. 10 schematically illustrates examples of structures of unnaturalbase pairs which pair by forces other than hydrogen bonding (PICS:PICS,3MN:3MN, 7AI:7AI, Dipic:Py).

FIG. 11 is a graph of results of a negative selection method forsuppressor tRNAs, which shows the percentage of surviving cellscontaining one of three constructs, for a given amount of time based onthe suppression of two amber codons in the barnase gene introduced by avector, e.g., plasmid pSCB2. This plasmid encodes the barnase genecontaining two amber codons. Selections are carried out in GMML liquidmedium, and 20 mM of arabinose is used to induce barnase expression.Three constructs are indicated by the following: (1) a circle whichrepresents a control plasmid with no suppressor tRNA; (2) a trianglewhich represents a suppressor tRNA on plasmid, pAC-YYG1; and, (3) asquare which represents a suppressor tRNA on plasmid, pAC-JY.

FIG. 12 displays growth histograms, illustrating positive selectionbased on the suppression of an amber codon in the ∃-lactamase gene. Avector encoding a suppressor tRNA, e.g., pAC plasmid, is cotransformedwith a vector encoding a synthetase, e.g., pBLAM-JYRS, in an organism,e.g., Escherichia coli DH10B cells. The growth of cells harboringsynthetase and different pAC plasmids in liquid 2×YT medium with variousconcentrations of ampicillin, e.g., 0, 100 and 500 μg/ml, is shown inPanel A, where pAC is a control plasmid with no suppressor tRNA, wherepAC-YYG1 is a plasmid with a suppressor tRNA, and where pAC-JY is aplasmid with a suppressor tRNA. Panel B shows positive selection of thesame constructs using 2×YT agar plates with 500 μg/ml ampicillin. Threeconstructs are indicated by the following: (1) a circle which representsa control plasmid with no suppressor tRNA; (2) a triangle whichrepresents a suppressor tRNA on plasmid, pAC-YYG1; and, (3) a squarewhich represents a suppressor tRNA on plasmid, pAC-JY.

FIG. 13 illustrates DNA sequences of mutant suppressor tRNAs selectedfrom anticodon-loop (AA2 (SEQ ID NO. 93), AA3 (SEQ ID NO. 94), and AA4(SEQ ID NO 95)) and all-loop library (J15 (SEQ ID NO. 96), J17 (SEQ IDNO. 97), J18 (SEQ ID NO. 98), J22 (SEQ ID NO. 99), N11 (SEQ ID NO. 100),N12 (SEQ ID NO. 101), N13 (SEQ ID NO. 102), and N16 (SEQ ID NO. 103)).JY stands for the wild-type Methanococcus jannaschii tRNACUATyrCUA (SEQID NO:92)

FIG. 14 schematically illustrates a stereo view of the active site ofTyrRS. Residues from B. stearothermophilus TyrRS are illustrated in thefigure. Corresponding residues from Methanococcus jannaschii TyrRS areTyr³²(Tyr³⁴), Ty Glu¹⁰⁷ (Asn¹²³), Asp⁵⁸(Asp₁₇₆, Ile¹⁵⁹ (Phe¹⁷⁷), andLeu¹⁶²(Leu¹⁸⁰) with residues from B. stearothermophilus TyrRS inparentheses.

FIG. 15 schematically illustrates a view of the active site of TyrRS.Residues from B. stearothermophilus TyrRS are illustrated in the figure.Corresponding residues from Methanococcus jannaschii TyrRS areTyr³²(Tyr³⁴), Asp¹⁵⁸(Asp¹⁷⁶), Ile¹⁵⁹(Phe¹⁷⁷), Leu¹⁶²(Leu¹⁸⁰) andAla¹⁶⁷(Gln¹⁸⁹) with residues from B. stearothermophilus TyrRS inparentheses.

FIG. 16, Panel A and Panel B schematically illustrate an example ofFACS-based selection and screening methods used to generate a componentof the present invention, e.g., orthogonal synthetase. Panel Aschematically illustrates vectors, e.g., plasmids, for expression oforthogonal synthetase library and O-tRNA (library plasmid) and for theT7 RNA polymerase/GFP reporter system (reporter plasmid), with one ormore selector codons, e.g., TAG. Panel B schematically illustratespositive selection/negative screen scheme, where the cells are grown thepresence and absence of the unnatural amino acid, the presence andabsence of a selection agent, and screened for fluorescing cells andnon-fluorescing cells in the screening process, where the “+” and emptycircles correspond to fluorescing and non-fluorescing cells,respectively.

FIG. 17, Panel A, Panel B, Panel C and Panel D illustrates anamplifiable fluorescence reporter system. Panel A schematicallyillustrates vectors that can be used in the screen, e.g., plasmids, suchas pREP, where T7 RNA polymerase transcription is controlled by the arapromoter; protein expression depends on suppression of amber codons atvarying locations in the gene. Reporter expression, e.g., GFPuvexpression is controlled by T7 RNA polymerase. The reporter vector,e.g., plasmid pREP, is compatible for use with a vector for expressingan orthogonal synthetase/tRNA pair, e.g., a ColE1 plasmid. Panel Billustrates compositions and fluorescence enhancement of T7 RNApolymerase gene constructs within pREP (1-12). The construct number isindicated to the left of each. Fluorescence enhancements, indicated tothe right of each construct, are calculated as the cellconcentration-corrected ratio of fluorescence, as measuredfluorimetrically, of cells containing pREP(1-12) and pQ or pQD. Theposition of the amber mutations within a gene are indicated. Panel Cillustrates cytometric analysis of cells containing pREP (10) and eitherpQD (top) or pQ (bottom). Panel D illustrates fluorimetric analyses ofcells containing pREP (10) and expressing various

Escherichia coli suppressor tRNAs. “None” indicates that the cellscontain no suppressor tRNA.

FIG. 18 schematically illustrates phage-based selection for theincorporation of unnatural amino acids into a surface epitope. Forexample, Escherichia coli carrying the mutant synthetase library areinfected by phage with a stop codon in a gene encoding a surfaceprotein. Phage containing an active synthetase display the unnaturalamino acid on the phage surface and are selected with immobilizedmonoclonal antibodies.

FIG. 19 schematically illustrates an example of a molecule, e.g.,immobilized aminoalkyl adenylate analog of the aminoacyl adenylateintermediate, used to screen displayed synthetases, e.g.,phage-displayed synthetases, with unnatural amino acid specificity.

FIG. 20 is a graph illustrating ampicillin resistance of variousorthogonal pairs from a variety of organisms. The figure illustrates anexample of finding an orthogonal pair using a reporter constructs, eachcontaining a reporter gene, e.g., a β-lactamase gene, with a selectorcodon, e.g., an amber codon, and a suppressor tRNA (with a selectoranticodon), where the suppressor tRNA can be from a variety oforganisms, e.g., A. fulgidus, Halobacterium NRC-1, P. furiosus, P.horikoshii, and Methanococcus jannaschii. The reporter constructs andcloned synthetases from different organisms, e.g., M.thermoautotrophicum, Methanococcus jannaschii, P. horikoshii, A. pernix,A. fulgidus, Halobacterium NRC-1, and Escherichia coli are transformedinto a cell. Cells are grown in various concentrations of a selectoragent, e.g., ampicillin. Cells possessing an orthogonal tRNA/RS pair areselected, e.g., using an in vivo complementation assay. As shown, twosystems showed suppression levels significant higher than was observedwith Escherichia coli synthetase. They are M. thermoautotrophicum andMethanococcus jannaschii.

FIG. 21, Panel A and Panel B, illustrates mutated amber suppressor tRNAsfrom a Halobacterium NRC-1, which are generated by mutating, e.g.,randomizing, the anticodon loop of the leucyl tRNA (SEQ ID NO:104) andselecting (Panel B) for more efficient suppression of a selector codon,e.g., an amber codon in a reporter gene(s), e.g., using a combination ofselection steps, such as selection based on β-lactamase and selectionbased on barnase. Panel B illustrates IC50 values in μg/ml of ampicillinfor a β-lactamase amber suppression system with three mutant tRNAconstructs, original amber mutant, optimized anticodon loop, andoptimized acceptor stem, alone or with an RS, e.g., MtLRS. The optimizedanticodon and optimized acceptor stem gave the highest values in theβ-lactamase selection step.

FIG. 22 illustrates a tRNA suppressor for a base codon (SEQ ID NO:105).The tRNA suppressor illustrated in this figure was isolated from alibrary derived from the Halobacterium NRC-1 TTG tRNA, where theanticodon loop was randomized with 8 nucleotides and subjected toampicillin selection with a reporter construct containing a β-lactamasegene with an AGGA codon at the A184 site.

FIG. 23 Panels A-D, illustrates the activity of the dominant synthetasevariant from each successful evolution experiment. FIG. 23A is aphotograph illustrating long-wavelength ultraviolet illumination ofcells containing pREP/YC-JYCUA and the indicated synthetase variant,grown in either the presence (+) or absence (−) of the correspondingunnatural amino acid. FIG. 23B illustrates a fluorimetric analysis ofcells containing pREP/YC-JYCUA and the indicated synthetase variant,grown in either the presence (left) or absence (right) of thecorresponding unnatural amino acid. FIG. 23C is a table that illustratesa Cm IC₅₀ analysis of cells containing pREP/YC-JYCUA and the indicatedsynthetase variant, grown in either the presence or absence of thecorresponding unnatural amino acid. FIG. 23D illustrates a proteinexpression analysis from cells containing pBAD/JYAMB-4TAG and theindicated synthetase variant, grown in either the presence (+) orabsence (−) of the corresponding unnatural amino acid.

FIG. 24, illustrates activity comparisons of OAY-RS variants derivedusing a negative FACS-based screen (OAY-RS(1,3,5)) or negativebarnase-based selection (OAY-RS(B)). Cells containing pREP/YC-JYCUA andthe indicated synthetase variant were grown in either the presence(solid block, left) or absence (solid block, right) of the correspondingunnatural amino acid and analyzed fluorimetrically. Fluorescenceenhancement (bar, back) is calculated as the cellconcentration-corrected ratio of fluorescence of cells grown in thepresence versus the absence of unnatural amino acid.

FIG. 25, Panels A-B, illustrate components of the multipurpose reporterplasmid system for directing the evolution of M. jannaschii TyrRS. FIG.25A illustrates plasmid pREP/YC-JYCUA. Plasmid pREP/YC-JYCUA iscompatible for use with plasmid pBK and variants. FIG. 25B illustratesstructures of unnatural amino acids used as targets for the evolution ofM. jannaschii TyrRS.

FIG. 26 illustrates the strategy for the evolution of an aminoacyl-tRNAsynthetase using plasmid pREP/YC-JYCUA. Fluorescent and non-fluorescentcells are shown in black and white, respectively.

FIG. 27 illustrates a threonyl-tRNA synthetase from Thermusthermophilus.

FIG. 28 illustrates the generation of an orthogonal tRNA for a Tthermophilus orthogonal threonyl-tRNA/RS. Wild type (SEQ ID NO:106) andtwo mutant (C₂G₇₃→A₂U₇₁ (SEQ ID NO. 107) and C₂G₇₁→A₂U₇₁G₃₄G₃₅U₃₆→C₃₄G₃₅U₃₆ (SEQ ID NO. 108)) tRNAs are illustrated.

FIG. 29 illustrates exemplary unnatrual amino acids as utilized in thecurrent invention.

FIG. 30 illustrates exemplary unnatrual amino acids as utilized in thecurrent invention.

FIG. 31 illustrates exemplary unnatrual amino acids as utilized in thecurrent invention.

DETAILED DESCRIPTION Introduction

Proteins are at the crossroads of virtually every biological process,from photosynthesis and vision to signal transduction and the immuneresponse. These complex functions result from a polyamide based polymerconsisting of twenty relatively simple building blocks arranged in adefined primary sequence.

The present invention includes methods and composition for use in thesite-specific incorporation of unnatural amino acids directly intoproteins in vivo. Importantly, the unnatural amino acid is added to thegenetic repertoire, rather than substituting for one of the common 20amino acids. The present invention provides methods for generating,methods for identifying and compositions comprising the components usedby the biosynthetic machinery to incorporate an unnatural amino acidinto a protein. The present invention, e.g., (i) allows thesite-selective insertion of one or more unnatural amino acids at anydesired position of any protein, (ii) is applicable to both prokaryoticand eukaryotic cells, (iii) enables in vivo studies of mutant proteinsin addition to the generation of large quantities of purified mutantproteins, and (iv) is adaptable to incorporate any of a large variety ofnon-natural amino acids, into proteins in vivo. Thus, in a specificpolypeptide sequence a number of different site-selective insertions ofunnatural amino acids is possible. Such insertions are optionally all ofthe same type (e.g., multiple examples of one type of unnatural aminoacid inserted at multiple points in a polypeptide) or are optionally ofdiverse types (e.g., different unnatural amino acid types are insertedat multiple points in a polypeptide).

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor biological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “amolecule” optionally includes a combination of two or more suchmolecules, and the like.

Unless defined otherwise, all scientific and technical terms areunderstood to have the same meaning as commonly used in the art to whichthey pertain. For the purpose of the present invention, the followingterms are defined below.

As used herein, proteins and/or protein sequences are “homologous” whenthey are derived, naturally or artificially, from a common ancestralprotein or protein sequence. Similarly, nucleic acids and/or nucleicacid sequences are homologous when they are derived, naturally orartificially, from a common ancestral nucleic acid or nucleic acidsequence. For example, any naturally occurring nucleic acid can bemodified by any available mutagenesis method to include one or moreselector codon. When expressed, this mutagenized nucleic acid encodes apolypeptide comprising one or more unnatural amino acid. The mutationprocess can, of course, additionally alter one or more standard codon,thereby changing one or more standard amino acid in the resulting mutantprotein as well. Homology is generally inferred from sequence similaritybetween two or more nucleic acids or proteins (or sequences thereof).The precise percentage of similarity between sequences that is useful inestablishing homology varies with the nucleic acid and protein at issue,but as little as 25% sequence similarity is routinely used to establishhomology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establishhomology. Methods for determining sequence similarity percentages (e.g.,BLASTP and BLASTN using default parameters) are described herein and aregenerally available.

The term “preferentially aminoacylates” refers to an efficiency, e.g.,about 70% efficient, about 75% efficient, about 85% efficient, about90%, about 95%, about 99% or more efficient, at which an O-RSaminoacylates an O-tRNA with an unnatural amino acid compared to anaturally occurring tRNA or starting material used to generate theO-tRNA. The unnatural amino acid is then incorporated into a growingpolypeptide chain with high fidelity, e.g., at greater than about 75%efficiency for a given selector codon, at greater than about 80%efficiency for a given selector codon, at greater than about 90%efficiency for a given selector codon, greater than about 95% efficiencyfor a given selector codon, or greater than about 99% efficiency for agiven selector codon.

The term “selector codon” refers to codons recognized by the O-tRNA inthe translation process and not recognized by an endogenous tRNA. TheO-tRNA anticodon loop recognizes the selector codon on the mRNA andincorporates its amino acid, e.g., an unnatural amino acid, at this sitein the polypeptide. Selector codons can include, e.g., nonsense codons,such as, stop codons, e.g., amber, ochre, and opal codons; four or morebase codons; codons derived from natural or unnatural base pairs and thelike. For a given system, a selector codon can also include one of thenatural three base codons, wherein the endogenous system does not usesaid natural three base codon, e.g., a system that is lacking a tRNAthat recognizes the natural three base codon or a system wherein thenatural three base codon is a rare codon.

As used herein, the term “orthogonal” refers to a molecule (e.g., anorthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNA synthetase(O-RS)) that is used with reduced efficiency by a system of interest(e.g., a translational system, e.g., a cell). Orthogonal refers to theinability or reduced efficiency, e.g., less than 20% efficient, lessthan 10% efficient, less than 5% efficient, or e.g., less than 1%efficient, of an orthogonal tRNA and/or orthogonal RS to function in thetranslation system of interest. For example, an orthogonal tRNA in atranslation system of interest is aminoacylated by any endogenous RS ofa translation system of interest with reduced or even zero efficiency,when compared to aminoacylation of an endogenous tRNA by the endogenousRS. In another example, an orthogonal RS aminoacylates any endogenoustRNA in the translation system of interest with reduced or even zeroefficiency, as compared to aminoacylation of the endogenous tRNA by anendogenous RS. “Improvement in orthogonality” refers to enhancedorthogonality compared to a starting material or a naturally occurringtRNA or RS.

The term “complementary” refers to components of an orthogonal pair,O-tRNA and O-RS that can function together, e.g., the O-RS aminoacylatesthe O-tRNA.

The term “derived from” refers to a component that is isolated from anorganism or isolated and modified, or generated, e.g., chemicallysynthesized, using information of the component from the organism.

The term “translation system” refers to the components necessary toincorporate a naturally occurring amino acid into a growing polypeptidechain (protein). For example, components can include ribosomes, tRNAs,synthetases, mRNA and the like. The components of the present inventioncan be added to a translation system, in vivo or in vitro.

The term “inactive RS” refers to a synthetase that have been mutated sothat it no longer can aminoacylate its cognate tRNA with an amino acid.

The term “selection agent” refers to an agent that when present allowsfor a selection of certain components from a population, e.g., anantibiotic, wavelength of light, an antibody, a nutrient or the like.The selection agent can be varied, e.g., such as concentration,intensity, etc.

The term “positive selection marker” refers to a marker that whenpresent, e.g., expressed, activated or the like, results inidentification of an organism with the positive selection marker fromthose without the positive selection marker.

The term “negative selection marker” refers to a marker that whenpresent, e.g., expressed, activated or the like, allows identificationof an organism that does not possess the desired property (e.g., ascompared to an organism which does possess the desired property).

The term “reporter” refers to a component that can be used to selectcomponents described in the present invention. For example, a reportercan include a green fluorescent protein, a firefly luciferase protein,or genes such as β-gal/lacZ (β-galactosidase), Adh (alcoholdehydrogenase) or the like.

The term “not efficiently recognized” refers to an efficiency, e.g.,less than about 10%, less than about 5%, or less than about 1%, at whicha RS from one organism aminoacylates O-tRNA.

The term “eukaryote” refers to organisms belonging to the phylogeneticdomain Eucarya such as animals (e.g., mammals, insects, reptiles, birds,etc.), ciliates, plants, fungi (e.g., yeasts, etc.), flagellates,microsporidia, protists, etc. Additionally, the term “prokaryote” refersto non-eukaryotic organisms belonging to the Eubacteria (e.g.,Escherichia coli, Thermus thermophilus, etc.) and Archaea (e.g.,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium such as Haloferax volcanii and Halobacterium speciesNRC-1, A. fulgidus, P. furiosus, P. horikoshii, A. pernix, etc.)phylogenetic domains

A “suppressor tRNA” is a tRNA that alters the reading of a messenger RNA(mRNA) in a given translation system. A suppressor tRNA can readthrough, e.g., a stop codon, a four base codon, or a rare codon.

Discussion

The present invention relates to methods and compositions for newcomponents of biosynthetic translational machinery that allows for theincorporation of unnatural amino acids into proteins in vivo.Specifically, compositions comprising and methods for generatingorthogonal tRNAs and orthogonal-RS and orthogonal tRNAs/orthogonal-RSpairs are provided. These components, when introduced into a host cell,can be used in the translation system of the cell to incorporate anunnatural amino acid in vivo into a polypeptide (protein) of interest.For example, this can provide site-specific unnatural amino acidmutagenesis; or, optionally, random unnatural amino acid mutagenesis.The orthogonal tRNA delivers the unnatural amino acid in response to aselector codon and the orthogonal synthetase preferentiallyaminoacylates an orthogonal tRNA with the unnatural amino acid. The O-RSdoes not efficiently aminoacylate the orthogonal tRNA with any of thecommon twenty amino acids. Methods for making and identifying orthogonalpairs are also provided.

The site-specific incorporation of unnatural amino acids into proteinsin vivo is schematically illustrated in FIG. 1. A selector codon, e.g.,a unique codon, is introduced into a gene of interest. The gene istranscribed into mRNA and conventional translation begins on theribosome. Endogenous synthetases aminoacylate endogenous tRNAs withnatural amino acids (aa) in the presence of ATP. An orthogonal tRNA isenzymatically aminoacylated by an orthogonal synthetase with anunnatural amino acid in the presence of ATP. When the ribosomeencounters a selector codon, an orthogonal tRNA, which is modified tocontain a selector anticodon, e.g., a unique anticodon, it is able todecode the mutation as an unnatural amino acid, and translation proceedsto the full-length product with the incorporated unnatural amino acid.

Orthogonal Aminoacyl tRNA Synthetase, O-RS

In order to specifically incorporate an unnatural amino acid in vivo,the substrate specificity of the synthetase is altered so that only thedesired unnatural amino acid, but not any common 20 amino acids arecharged to the tRNA. If the orthogonal synthetase is promiscuous, itwill result in mutant proteins with a mixture of natural and unnaturalamino acids at the target position. For instance, in an attempt tosite-specifically incorporate p-F-Phe, a yeast amber suppressortRNAPheCUA/phenylalanyl-tRNA synthetase pair was used in a p-F-Pheresistant, Phe auxotrophic Escherichia coli strain. See, e.g., R.Furter, Protein Sci., 7:419 (1998). Because yeast PheRS does not havehigh substrate specificity for p-F-Phe, the mutagenesis site wastranslated with 64-75% p-F-Phe and the remainder as Phe and Lys even inthe excess of p-F-Phe added to the growth media. In addition, at the Phecodon positions, 7% p-F-Phe was found, indicating that the endogenousEscherichia coli PheRS incorporates p-F-Phe in addition to Phe. Becauseof its translational infidelity, this approach is not generallyapplicable to other unnatural amino acids. Modification of the substratespecificity of a synthetase was expected to be difficult due to the highintrinsic fidelity of the natural synthetases and the fact thatunnatural amino acids are not required for any cellular function. Thepresent invention solves this problem and provides composition of, andmethods for, generating synthetases that have modified substratespecificity, such as an unnatural amino acid. Using the components ofthe present invention, the efficiency of incorporation of an unnaturalamino acid into is, e.g., greater than about 75%, greater than about85%, greater than about 95%, greater than about 99% or more.

Compositions of the present invention include an orthogonalaminoacyl-tRNA synthetase (O-RS), where the O-RS preferentiallyaminoacylates an orthogonal tRNA (O-tRNA) with an unnatural amino acid,optionally, in vivo. In one embodiment, the O-RS comprises a nucleicacid comprising a polynucleotide sequence selected from the groupconsisting of: SEQ ID NO: 4-34 (see, Table 5) and a complementarypolynucleotide sequence thereof. In another embodiment, the O-RS hasimproved or enhanced enzymatic properties, e.g., the K_(m) is lower, thek_(cat) is higher, the value of k_(cat)/K_(m) is higher or the like, forthe unnatural amino acid compared to a naturally occurring amino acid,e.g., one of the 20 known amino acids. Sequences of exemplary O-tRNA andO-RS molecules can be found in Example 10.

Methods for producing an O-RS are based on generating a pool of mutantsynthetases from the framework of a wild-type synthetase, and thenselecting for mutated RSs based on their specificity for an unnaturalamino acid relative to the common twenty. To isolate such a synthetase,the selection methods of the present invention are: (i) sensitive, asthe activity of desired synthetases from the initial rounds can be lowand the population small; (ii) “tunable”, since it is desirable to varythe selection stringency at different selection rounds; and, (iii)general, so that it can be used for different unnatural amino acids.

The present invention provides methods to generate an orthogonalaminoacyl tRNA synthetase by mutating the synthetase, e.g., at theactive site in the synthetase, at the editing mechanism site in thesynthetase, at different sites by combining different domains ofsynthetases, or the like, and applying a selection process. FIG. 2,Panel A schematically illustrates an in vivo selection/screen strategy,which is based on the combination of a positive selection followed by anegative selection. In the positive selection, suppression of theselector codon introduced at a nonessential position(s) of a positivemarker allows cells to survive under positive selection pressure. In thepresence of both natural and unnatural amino acids, survivors thusencode active synthetases charging the orthogonal suppressor tRNA witheither a natural or unnatural amino acid. In the negative selection,suppression of a selector codon introduced at a nonessential position(s)of a negative marker removes synthetases with natural amino acidspecificities. Survivors of the negative and positive selection encodesynthetases that aminoacylate (charge) the orthogonal suppressor tRNAwith unnatural amino acids only. These synthetases can then be subjectedto further mutagenesis, e.g., DNA shuffling or other recursivemutagenesis methods. Of course, in other embodiments, the inventionoptionall cn utilize different orders of steps to identify (e.g., O-RS,O-tRNA, pairs, etc.), e.t., negative selection/screening followed bypositive selection/screening or vice verse or any such combinationsthereof.

For example, see, FIG. 2, Panel B. In FIG. 2, Panel B, a selector codon,e.g., an amber codon, is placed in a reporter gene, e.g., an antibioticresistance gene, such as β-lactamase, with a selector codon, e.g., TAG.This is placed in an expression vector with members of the mutated RSlibrary. This expression vector along with an expression vector with anorthogonal tRNA, e.g., a orthogonal suppressor tRNA, are introduced intoa cell, which is grown in the presence of a selection agent, e.g.,antibiotic media, such as ampicillin. Only if the synthetase is capableof aminoacylating (charging) the suppressor tRNA with some amino aciddoes the selector codon get decoded allowing survival of the cell onantibiotic media.

Applying this selection in the presence of the unnatural amino acid, thesynthetase genes that encode synthetases that have some ability toaminoacylate are selected away from those synthetases that have noactivity. The resulting pool of synthetases can be charging any of the20 naturally occurring amino acids or the unnatural amino acid. Tofurther select for those synthetases that exclusively charge theunnatural amino acid, a second selection, e.g., a negative selection, isapplied. In this case, an expression vector containing a negativeselection marker and an O-tRNA is used, along with an expression vectorcontaining a member of the mutated RS library. This negative selectionmarker contains at least one selector codon, e.g., TAG. These expressionvectors are introduced into another cell and grown without unnaturalamino acids and, optionally, a selection agent, e.g., tetracycline. Inthe negative selection, those synthetases with specificities for naturalamino acids charge the orthogonal tRNA, resulting in suppression of aselector codon in the negative marker and cell death. Since no unnaturalamino acid is added, synthetases with specificities for the unnaturalamino acid survive. For example, a selector codon, e.g., a stop codon,is introduced into the reporter gene, e.g., a gene that encodes a toxicprotein, such as barnase. If the synthetase is able to charge thesuppressor tRNA in the absence of unnatural amino acid, the cell will bekilled by translating the toxic gene product. Survivors passing bothselection/screens encode synthetases specifically charging theorthogonal tRNA with an unnatural amino acid.

In one embodiment, methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O-RS) include: (a) generating alibrary of mutant RSs derived from at least one aminoacyl-tRNAsynthetase (RS) from a first organism; (b) selecting the library ofmutant RSs for members that aminoacylate an orthogonal tRNA (O-tRNA) inthe presence of an unnatural amino acid and a natural amino acid,thereby providing a pool of active mutant RSs; and, (c) negativelyselecting the pool for active mutant RSs that preferentiallyaminoacylate the O-tRNA in the absence of the unnatural amino acid,thereby providing the at least one recombinant O-RS; wherein the atleast one recombinant O-RS preferentially aminoacylates the O-tRNA withthe unnatural amino acid. Optionally, more mutations are introduced bymutagenesis, e.g., random mutagenesis, recombination or the like, intothe selected synthetase genes to generate a second-generation synthetaselibrary, which is used for further rounds of selection until a mutantsynthetase with desired activity is evolved. Recombinant O-RSs producedby the methods are included in the present invention. As explainedbelow, orthogonal tRNA/synthetase pairs or the invention are alsooptionally generated by importing such from a first organism into asecond organism.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 5 amino acids to different aminoacids, e.g., alanine.

The library of mutant RSs can be generated using various mutagenesistechniques known in the art. For example, the mutant RSs can begenerated by site-specific mutations, random point mutations, in vitrohomologous recombinant, chimeric constructs or the like. In oneembodiment, mutations are introduced into the editing site of thesynthetase to hamper the editing mechanism and/or to alter substratespecificity. See, e.g., FIG. 3 and FIG. 4. FIG. 3 illustratessite-specific mutations to generate directed libraries for tyrosineanalogues. FIG. 4 illustrates a consensus sequence forpentafluorophenylalanine selection to generate directed libraries forthese analogues. Libraries of mutant RSs also include chimericsynthetase libraries, e.g., libraries of chimeric Methanococcusjannaschii/Escherichia coli synthetases. The domain of one synthetasecan be added or exchanged with a domain from another synthetase. FIG. 5schematically illustrates the transplantation of one domain, e.g., theCPI domain, from one organism, e.g., Escherichia coli, to the synthetaseof other organism, e.g., Methanococcus jannaschii TyrRS. CPI can betransplanted from Escherichia coli TyrRS to H. sapiens TyrRS. See, e.g.,Wakasugi, K., et al., EMBO J. 17:297-305 (1998). FIG. 6 schematicallyillustrates the construction of chimeric Methanococcusjannaschii/Escherichia coli synthetases and FIG. 7 schematicallyillustrates the generation of a library of chimeric synthetases, e.g.,Methanococcus jannaschii/Escherichia coli synthetases. See, e.g.,Sieber, et al., Nature Biotechnology, 19:456-460 (2001). The chimericlibrary is screened for a variety of properties, e.g., for members thatare expressed and in frame, for members that lack activity with adesired synthetase, and/or for members that show activity with a desiredsynthetase.

In one embodiment, the positive selection step includes: introducing apositive selection marker, e.g., an antibiotic resistance gene, or thelike, and the library of mutant RSs into a plurality of cells, whereinthe positive selection marker comprises at least one selector codon,e.g., an amber codon; growing the plurality of cells in the presence ofa selection agent; selecting cells that survive in the presence of theselection agent by suppressing the at least one selector codon in thepositive selection marker, thereby providing a subset of positivelyselected cells that contains the pool of active mutant RSs. Optionally,the selection agent concentration can be varied.

In one embodiment, negative selection includes: introducing a negativeselection marker with the pool of active mutant RSs from the positiveselection into a plurality of cells of a second organism, wherein thenegative selection marker is an antibiotic resistance gene, e.g., achloramphenicol acetyltransferase (CAT) gene, comprising at least oneselector codon; and, selecting cells that survive in a 1st mediasupplemented with the unnatural amino acid and a selection agent, butfail to survive in a 2nd media not supplemented with the unnatural aminoacid and the selection agent, thereby providing surviving cells with theat least one recombinant O-RS. Optionally, the concentration of theselection agent is varied.

The 1^(st) and 2^(nd) media described above can include, e.g., a directreplica plate method. For example, after passing the positive selection,cells are grown in the presence of either ampicillin or chloramphenicoland the absence of the unnatural amino acid. Those cells that do notsurvive are isolated from a replica plate supplemented with theunnatural amino acid. No transformation into a second negative selectionstrain is needed, and the phenotype is known. Compared to otherpotential selection markers, a positive selection based on antibioticresistance offers the ability to tune selection stringency by varyingthe concentration of the antibiotic, and to compare the suppressionefficiency by monitoring the highest antibiotic concentration cells cansurvive. In addition, the growth process is also an enrichmentprocedure. This can lead to a quick accumulation of the desiredphenotype.

In another embodiment, negatively selecting the pool for active mutantRSs includes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection marker, wherein thenegative selection marker is a toxic marker gene, e.g., a ribonucleasebarnase gene, comprising at least one selector codon, and the pool ofactive mutant RSs into a plurality of cells of a second organism; andselecting cells that survive in a 1st media not supplemented with theunnatural amino acid, but fail to survive in a 2nd media supplementedwith the unnatural amino acid, thereby providing surviving cells withthe at least one recombinant O-RS, wherein the at least one recombinantO-RS is specific for the unnatural amino acid. Optionally, the negativeselection marker comprises two or more selector codons.

In one aspect, positive selection is based on suppression of a selectorcodon in a positive selection marker, e.g., a chloramphenicolacetyltransferase (CAT) gene comprising a selector codon, e.g., an amberstop codon, in the CAT gene, so that chloramphenicol can be applied asthe positive selection pressure. In addition, the CAT gene can be usedas both a positive marker and negative marker as describe herein in thepresence and absence of unnatural amino acid. Optionally, the CAT genecomprising a selector codon is used for the positive selection and anegative selection marker, e.g., a toxic marker, such as a barnase genecomprising at least one or more selector codons, is used for thenegative selection.

In another aspect, positive selection is based on suppression of aselector codon at nonessential position in the β-lactamase gene,rendering cells ampicillin resistant; and a negative selection using theribonuclease barnase as the negative marker is used. In contrast toβ-lactamase, which is secreted into the periplasm, CAT localizes in thecytoplasm; moreover, ampicillin is bacteriocidal, while chloramphenicolis bacteriostatic.

The recombinant O-RS can be further mutated and selected. In oneembodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O-RS) can further comprise: (d)isolating the at least one recombinant O-RS; (e) generating a second setof mutated O-RS derived from the at least one recombinant O-RS; and, (f)repeating steps (b) and (c) until a mutated O-RS is obtained thatcomprises an ability to preferentially aminoacylate the O-tRNA.Optionally, steps (d)-(f) are repeated, e.g., at least about two times.In one aspect, the second set of mutated O-RS can be generated bymutagenesis, e.g., random mutagenesis, site-specific mutagenesis,recombination or a combination thereof.

The stringency of the selection steps, e.g., the positive selection step(b), the negative selection step (c) or both the positive and negativeselection steps (b) and (c), in the above described-methods, optionallyinclude varying the selection stringency. For example, because barnaseis an extremely toxic protein, the stringency of the negative selectioncan be controlled by introducing different numbers of selector codonsinto the barnase gene. In one aspect of the present invention, thestringency is varied because the desired activity can be low duringearly rounds. Thus, less stringent selection criteria are applied inearly rounds and more stringent criteria are applied in later rounds ofselection.

Other types of selections can be used in the present invention for,e.g., O-RS, O-tRNA, and O-tRNA/O-RS pair. For example, the positiveselection step (b), the negative selection step (c) or both the positiveand negative selection steps (b) and (c) can include using a reporter,wherein the reporter is detected by fluorescence-activated cell sorting(FACS). For example, a positive selection can be done first with apositive selection marker, e.g., chloramphenicol acetyltransferase (CAT)gene, where the CAT gene comprises a selector codon, e.g., an amber stopcodon, in the CAT gene, which followed by a negative selection screen,that is based on the inability to suppress a selector codon(s), e.g.,two or more, at positions within a negative marker, e.g., T7 RNApolymerase gene. In one embodiment, the positive selection marker andthe negative selection marker can be found on the same vector, e.g.,plasmid. Expression of the negative marker drives expression of thereporter, e.g., green fluorescent protein (GFP). The stringency of theselection and screen can be varied, e.g., the intensity of the lightneed to fluorescence the reporter can be varied. In another embodiment,a positive selection can be done with a reporter as a positive selectionmarker, which is screened by FACs, followed by a negative selectionscreen, that is based on the inability to suppress a selector codon(s),e.g., two or more, at positions within a negative marker, e.g., barnasegene.

Optionally, the reporter is displayed on a cell surface, e.g., on aphage display or the like. Cell-surface display, e.g., the OmpA-basedcell-surface display system, relies on the expression of a particularepitope, e.g., a poliovirus C3 peptide fused to an outer membrane porinOmpA, on the surface of the Escherichia coli cell. The epitope isdisplayed on the cell surface only when a selector codon in the proteinmessage is suppressed during translation. The displayed peptide thencontains the amino acid recognized by one of the mutant aminoacyl-tRNAsynthetases in the library, and the cell containing the correspondingsynthetase gene can be isolated with antibodies raised against peptidescontaining specific unnatural amino acids. The OmpA-based cell-surfacedisplay system was developed and optimized by Georgiou et al. as analternative to phage display. See, Francisco, J. A., Campbell, R.,Iverson, B. L. & Georgoiu, G. Production and fluorescence-activated cellsorting of Escherichia coli expressing a functional antibody fragment onthe external surface. Proc. Natl. Acad. Sci. USA 90:10444-8 (1993).

Other embodiments of the present invention include carrying one or moreof the selection steps in vitro. The selected component, e.g.,synthetase and/or tRNA, can then be introduced into a cell for use in invivo incorporation of an unnatural amino acid.

Orthogonal tRNA

Compositions of an orthogonal tRNA (O-tRNA) are also a feature of theinvention, e.g., where the O-tRNA recognizes a selector codon and theO-tRNA is preferentially aminoacylated with an unnatural amino acid byan orthogonal aminoacyl-tRNA synthetase. In one embodiment, the O-tRNAcomprises a nucleic acid comprising a polynucleotide sequence selectedfrom the group consisting of: SEQ ID NO: 4-34 (see, Table 5) and acomplementary polynucleotide sequence thereof.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) areprovided herein. For example, to improve the orthogonality of a tRNAwhile preserving its affinity toward a desired RS, the methods include acombination of negative and positive selections with a mutant suppressortRNA library in the absence and presence of the cognate synthetase,respectively. See, FIG. 8. In the negative selection, a selectorcodon(s) is introduced in a marker gene, e.g., a toxic gene, such asbarnase, at a nonessential position. When a member of the mutated tRNAlibrary, e.g., derived from Methanococcus jannaschii, is aminoacylatedby endogenous host, e.g., Escherichia coli synthetases (i.e., it is notorthogonal to the host, e.g., Escherichia coli synthetases), theselector codon, e.g., an amber codon, is suppressed and the toxic geneproduct produced leads to cell death. Cells harboring orthogonal tRNAsor non-functional tRNAs survive. Survivors are then subjected to apositive selection in which a selector codon, e.g., an amber codon, isplaced in a positive marker gene, e.g., a drug resistance gene, such aβ-lactamase gene. These cells also contain an expression vector with acognate RS. These cells are grown in the presence of a selection agent,e.g., ampicillin. tRNAs are then selected for their ability to beaminoacylated by the coexpressed cognate synthetase and to insert anamino acid in response to this selector codon. Cells harboringnon-functional tRNAs, or tRNAs that cannot be recognized by thesynthetase of interest are sensitive to the antibiotic. Therefore, tRNAsthat: (i) are not substrates for endogenous host, e.g., Escherichiacoli, synthetases; (ii) can be aminoacylated by the synthetase ofinterest; and (iii) are functional in translation survive bothselections.

Methods of producing a recombinant O-tRNA include: (a) generating alibrary of mutant tRNAs derived from at least one tRNA, e.g., asuppressor tRNA, from a first organism; (b) negatively selecting thelibrary for mutant tRNAs that are aminoacylated by an aminoacyl-tRNAsynthetase (RS) from a second organism in the absence of a RS from thefirst organism, thereby providing a pool of mutant tRNAs; and, (c)selecting the pool of mutant tRNAs for members that are aminoacylated byan introduced orthogonal RS (O-RS), thereby providing at least onerecombinant O-tRNA; wherein the at least one recombinant O-tRNArecognizes a selector codon and is not efficiency recognized by the RSfrom the second organism and is preferentially aminoacylated by theO-RS. In one embodiment, the recombinant O-tRNA possesses an improvementof orthogonality.

Libraries of mutated tRNA are constructed. See, for example, FIG. 9.Mutations can be introduced at a specific position(s), e.g., at anonconservative position(s), or at a conservative position, at arandomized position(s), or a combination of both in a desired loop of atRNA, e.g., an anticodon loop, (D arm, V loop, TPC arm) or a combinationof loops or all loops. Chimeric libraries of tRNA are also included inthe present invention. It should be noted that libraries of tRNAsynthetases from various organism (e.g., microorganisms such aseubacteria or archaebacteria) such as libraries comprising naturaldiversity (such as libraries that comprise natural diversity (see, e.g.,U.S. Pat. No. 6,238,884 to Short et al. and references therein, U.S.Pat. No. 5,756,316 to Schallenberger et al; U.S. Pat. No. 5,783,431 toPetersen et al; U.S. Pat. No. 5,824,485 to Thompson et al; and U.S. Pat.No. 5,958,672 to Short et al), are optionally constructed and screenedfor orthogonal pairs.

In one embodiment, negatively selecting the library for mutant tRNAsthat are aminoacylated by an aminoacyl-tRNA synthetase (step (b) above)includes: introducing a toxic marker gene, wherein the toxic marker genecomprises at least one of the selector codons and the library of mutanttRNAs into a plurality of cells from the second organism; and, selectingsurviving cells, wherein the surviving cells contain the pool of mutanttRNAs comprising at least one orthogonal tRNA or nonfunctional tRNA. Forexample, the toxic marker gene is optionally a ribonuclease barnasegene, wherein the ribonuclease barnase gene comprises at least one ambercodon. Optionally, the ribonuclease barnase gene can include two or moreamber codons. The surviving cells can be selected, e.g., by using acomparison ratio cell density assay.

In one embodiment, selecting the pool of mutant tRNAs for members thatare aminoacylated by an introduced orthogonal RS (O-RS) can include:introducing a positive selection marker gene, wherein the positiveselection marker gene comprises a drug resistance gene, e.g., a∃-lactamase gene, comprising at least one of the selector codons, e.g.,a ∃-lactamase gene comprising at least one amber stop codon, the O-RS,and the pool of mutant tRNAs into a plurality of cells from the secondorganism; and, selecting surviving cells grown in the presence of aselection agent, e.g., an antibiotic, thereby providing a pool of cellspossessing the at least one recombinant tRNA, wherein the recombinanttRNA is aminoacylated by the O-RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection agent is varied. Recombinant O-tRNAsproduced by the methods are included in the present invention.

As described above for generating O-RS, the stringency of the selectionsteps can be varied. In addition, other selection/screening procedures,which are described herein, such as FACs, cell and phage display canalso be used.

Selector Codons

Selector codons of the present invention expand the genetic codonframework of protein biosynthetic machinery. For example, a selectorcodon includes, e.g., a unique three base codon, a nonsense codon, suchas a stop codon, e.g., an amber codon, or an opal codon, an unnaturalcodon, a four (or more) base codon or the like. A number of selectorcodons can be introduced into a desired gene, e.g., one or more, two ormore, more than three, etc. Additionally, it will be appreciated thatmultiple different (or similar or identical) unnatural amino acids canthus be incorporated precisely into amino acids (i.e., through use ofthe multiple selector codons).

The 64 genetic codons code for 20 amino acids and 3 stop codons. Becauseonly one stop codon is needed for translational termination, the othertwo can in principle be used to encode nonproteinogenic amino acids. Theamber stop codon, UAG, has been successfully used in in vitrobiosynthetic system and in Xenopus oocytes to direct the incorporationof unnatural amino acids. Among the 3 stop codons, UAG is the least usedstop codon in Escherichia coli. Some Escherichia coli strains containnatural suppressor tRNAs, which recognize UAG and insert a natural aminoacid in response to UAG. In addition, these amber suppressor tRNAs havebeen widely used in conventional protein mutagenesis. Different speciespreferentially use different codons for their natural amino acids, suchpreferentiallity is optionally utilized in designing/choosing theselector codons herein.

Although discussed with reference to unnatural amino acids herein, itwill be appreciated that a similar strategy can be used incorporate anatural amino acid in response to a particular selector codon. That is,a synthetase can be modified to load a natural amino acid onto anorthogonal tRNA that recognizes a selector codon in a manner similar tothe loading of an unnatural amino acid as described throughout.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of unnatural amino acids in vivo.For example, an O-tRNA is generated that recognizes the stop codon,e.g., UAG, and is aminoacylated by an O-RS with a desired unnaturalamino acid. This O-tRNA is not recognized by the naturally occurringaminoacyl-tRNA synthetases. Conventional site-directed mutagenesis canbe used to introduce the stop codon, e.g., TAG, at the site of interestin the protein gene. See, e.g., Sayers, J. R., Schmidt, W. Eckstein, F.5′, 3′ Exonuclease in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res, 791-802 (1988). When the O-RS, O-tRNAand the mutant gene are combined in vivo, the unnatural amino acid isincorporated in response to the UAG codon to give a protein containingthe unnatural amino acid at the specified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the host, e.g., Escherichia coli. Forexample, because the suppression efficiency for the UAG codon dependsupon the competition between the O-tRNA, e.g., the amber suppressortRNA, and the release factor 1 (RF1) (which binds to the UAG codon andinitiates release of the growing peptide from the ribosome), thesuppression efficiency can be modulated by, e.g., either increasing theexpression level of O-tRNA, e.g., the suppressor tRNA, or using an RF1deficient strain. Additionally, suppression efficiency and unnaturalamino acid uptake by carrying out random mutagenesis on an organism oron a portion of an organism's genome and performing proper selectionusing, e.g., one of the reporter systems described herein.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., C. H. Ma, W. Kudlicki, 0. W. Odom, G. Kramer and B.Hardesty, Biochemistry, 32:7939 (1993). In this case, the synthetic tRNAcompetes with the naturally occurring tRNA^(Arg), which exists as aminor species in Escherichia coli. Some organisms do not use all tripletcodons. An unassigned codon AGA in Micrococcus luteus has been utilizedfor insertion of amino acids in an in vitro transcription/translationextract. See, e.g., A. K. Kowal and J. S. Oliver, Nucl. Acid. Res.,25:4685 (1997). Components of the present invention can be generated touse these rare codons in vivo.

Selector codons also comprise four or more base codons, such as, four,five six or more. Examples of four base codons include, e.g., AGGA,CUAG, UAGA, CCCU and the like. Examples of five base codons include,e.g., AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. Forexample, in the presence of mutated O-tRNAs, e.g., a special frameshiftsuppressor tRNAs, with anticodon loops, e.g., with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, e.g., atleast a four-base codon, at least a five-base codon, or at least asix-base codon or more. Since there are 256 possible four-base codons,multiple unnatural amino acids can be encoded in the same cell using thefour or more base codon. See also, J. Christopher Anderson et al.,Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology,Vol. 9, 237-244 (2002); Thomas J. Magliery, Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769 (2001).

Methods of the present invention include using extended codons based onframeshift suppression. Four or more base codons can insert, e.g., oneor multiple unnatural amino acids into the same protein. For example,four-base codons have been used to incorporate unnatural amino acidsinto proteins using in vitro biosynthetic methods. See, e.g., C. H. Ma,W. Kudlicki, O. W. Odom, G. Kramer and B. Hardesty, Biochemistry, 1993,32, 7939 (1993); and, T. Hohsaka, D. Kajihara, Y. Ashizuka, H. Murakamiand M. Sisido, J. Am. Chem. Soc . . . 121:34 (1999). CGGG and AGGU wereused to simultaneously incorporate 2-naphthylalanine and an NBDderivative of lysine into streptavidin in vitro with two chemicallyacylated frameshift suppressor tRNAs. See, e.g., T. Hohsaka, Y.Ashizuka, H. Sasaki, H. Murakami and M. Sisido, J. Am. Chem. Soc.,121:12194 (1999). In an in vivo study, Moore et al. examined the abilityof tRNALeu derivatives with NCUA anticodons to suppress UAGN codons (Ncan be U, A, G, or C), and found that the quadruplet UAGA can be decodedby a tRNALeu with a UCUA anticodon with an efficiency of 13 to 26% withlittle decoding in the 0 or −1 frame. See, B. Moore, B. C. Persson, C.C. Nelson, R. F. Gesteland and J. F. Atkins, J. Mol. Biol., 298:195(2000). In one embodiment, extended codons based on rare codons ornonsense codons can be used in present invention, which can reducemissense readthrough and frameshift suppression at unwanted sites.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is inserted into a gene but is nottranslated into protein. The sequence contains a structure that servesas a cue to induce the ribosome to hop over the sequence and resumetranslation downstream of the insertion.

Alternatively, or in combination with others methods described above toincorporate an unnatural amino acid in a polypeptide, atrans-translation system can be used. This system involves a moleculecalled tmRNA present in Escherichia coli. This RNA molecule isstructurally related to an alanyl tRNA and is aminoacylated by thealanyl synthetase. The difference between tmRNA and tRNA is that theanticodon loop is replaced with a special large sequence. This sequenceallows the ribosome to resume translation on sequences that have stalledusing an open reading frame encoded within the tmRNA as template. In thepresent invention, an orthogonal tmRNA can be generated that ispreferentially aminoacylated with an orthogonal synthetase and loadedwith an unnatural amino acid. By transcribing a gene by the system, theribosome stalls at a specific site; the unnatural amino acid isintroduced at that site, and translation resumes using the sequenceencoded within the orthogonal tmRNA.

Selector codons also optionally include unnatural base pairs. Theseunnatural base pairs further expand the existing genetic alphabet. Oneextra base pair increases the number of triplet codons from 64 to 125.Properties of third base pairs include stable and selective basepairing, efficient enzymatic incorporation into DNA with high fidelityby a polymerase, and the efficient continued primer extension aftersynthesis of the nascent unnatural base pair. Descriptions of unnaturalbase pairs which can be adapted for methods and compositions include,e.g., Hirao, et al., An unnatural base pair for incorporating amino acidanalogues into protein, Nature Biotechnology, 20:177-182 (2002). Otherpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., C. Switzer, S. E. Moroney and S. A. Benner,J. Am. Chem. Soc., 111:8322 (1989); and, J. A. Piccirilli, T. Krauch, S.E. Moroney and S. A. Benner, Nature, 1990, 343:33 (1990); and E. T.Kool, Curr. Opin. Chem. Biol., 4:602 (2000). These bases in generalmispair to some degree with natural bases and cannot be enzymaticallyreplicated. Kool and co-workers demonstrated that hydrophobic packinginteractions between bases can replace hydrogen bonding to drive theformation of base pair. See, E. T. Kool, Curr. Opin. Chem. Biol., 4:602(2000); and, K. M. Guckian and E. T. Kool, Angew. Chem. Int. Ed. Engl.,36, 2825 (1998). In an effort to develop an unnatural base pairsatisfying all the above requirements, Schultz, Romesberg and co-workershave systematically synthesized and studied a series of unnaturalhydrophobic bases. The PICS:PICS self-pair, which is shown in FIG. 10,is found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by the Klenow fragment of Escherichiacoli DNA polymerase I (KF). See, e.g., D. L. McMinn, A. K. Ogawa, Y. Q.Wu, J. Q. Liu, P. G. Schultz and F. E. Romesberg, J. Am. Chem. Soc.,121:11586 (1999); and, A. K. Ogawa, Y. Q. Wu, D. L. McMinn, J. Q. Liu,P. G. Schultz and F. E. Romesberg, J. Am. Chem. Soc., 122:3274 (2000). A3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., A. K. Ogawa,Y. Q. Wu, M. Berger, P. G. Schultz and F. E. Romesberg, J. Am. Chem.Soc., 122:8803 (2000). However, both bases act as a chain terminator forfurther replication. A mutant DNA polymerase has been recently evolvedthat can be used to replicate the PICS self pair. In addition, a 7AIself pair can be replicated using a combination of KF and pol 3polymerase. See, e.g., E. J. L. Tae, Y. Q. Wu, G. Xia, P. G. Schultz andF. E. Romesberg, J. Am. Chem. Soc., 123:7439 (2001). A novel metallobasepair, Dipic:Py, has also been developed, which forms a stable pair uponbinding Cu(II). See, E. Meggers, P. L. Holland, W. B. Tolman, F. E.Romesberg and P. G. Schultz, J. Am. Chem. Soc., 122:10714 (2000).Because extended codons and unnatural codons are intrinsicallyorthogonal to natural codons, the methods of the present invention cantake advantage of this property to generate orthogonal tRNAs for them.

Orthogonal tRNA and Orthogonal Aminoacyl-tRNA Synthetase Pairs

An orthogonal pair is composed of an O-tRNA, e.g., a suppressor tRNA, aframeshift tRNA, or the like, and an O-RS. The O-tRNA is not acylated byendogenous synthetases and is capable of decoding a selector codon, asdescribed above. The O-RS recognizes the O-tRNA, e.g., with an extendedanticodon loop, and preferentially aminoacylates the O-tRNA with anunnatural amino acid. Methods for generating orthogonal pairs along withcompositions of orthogonal pairs are included in the present invention.The development of multiple orthogonal tRNA/synthetase pairs can allowthe simultaneous incorporation of multiple unnatural amino acids usingdifferent codons into the same polypeptide/protein.

In the present invention, methods and related compositions relate to thegeneration of orthogonal pairs (O-tRNA/O-RS) that can incorporate anunnatural amino acid into a protein in vivo. For example, compositionsof O-tRNAs of the present invention can comprise an orthogonalaminoacyl-tRNA synthetase (O-RS). In one embodiment, the O-tRNA and theO-RS can be complementary, e.g., an orthogonal O-tRNA/O-RS pair.Examples of pairs include a mutRNATyr-mutTyrRS pair, such as amutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like. In oneembodiment, an orthogonal pair of the present invention comprises thedesired properties of the orthogonal tRNA-aminoacyl-tRNA synthetase pairand is other than a mutRNAGIn-mutGlnRS derived from Escherichia coli, amutRNAAsp-mutAspRS derived from yeast or amutRNAPheCUA-mutphenlalanineRS from yeast, where these pairs do notpossess the properties of the pairs of the present invention.

The O-tRNA and the O-RS can be derived by mutation of a naturallyoccurring tRNA and/or RS from a variety of organisms, which aredescribed under sources and hosts. In one embodiment, the O-tRNA andO-RS are derived from at least one organism. In another embodiment, theO-tRNA is derived by mutation of a naturally occurring or mutatednaturally occurring tRNA from a first organism and the O-RS is derivedby mutation of a naturally occurring or mutated naturally occurring RSfrom a second organism.

Methods for generating specific O-tRNA/O-RS pairs are also provided inthe present invention. These methods solve the problems discussed belowfor the other strategies that were attempted to generate orthogonaltRNA/RS pairs. Specifically, methods of the present invention include:(a) generating a library of mutant tRNAs derived from at least one tRNAfrom a first organism; (b) negatively selecting the library for mutanttRNAs that are aminoacylated by an aminoacyl-tRNA synthetase (RS) from asecond organism in the absence of a RS from the first organism, therebyproviding a pool of mutant tRNAs; (c) selecting the pool of mutant tRNAsfor members that are aminoacylated by an introduced orthogonal RS(O-RS), thereby providing at least one recombinant O-tRNA. The at leastone recombinant O-tRNA recognizes a selector codon and is not efficiencyrecognized by the RS from the second organism and is preferentiallyaminoacylated by the O-RS. The method also includes: (d) generating alibrary of mutant RSs derived from at least one aminoacyl-tRNAsynthetase (RS) from a third organism; (e) selecting the library ofmutant RSs for members that preferentially aminoacylate the at least onerecombinant O-tRNA in the presence of an unnatural amino acid and anatural amino acid, thereby providing a pool of active mutant RSs; and,(f) negatively selecting the pool for active mutant RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the unnatural amino acid, thereby providing the at least onespecific O-tRNA/O-RS pair, where the at least one specific O-tRNA/O-RSpair comprises at least one recombinant O-RS that is specific for theunnatural amino acid and the at least one recombinant O-tRNA. Pairsproduced by the methods of the present invention are also included.

Previously, generation of an orthogonal tRNA/synthetase pair from anexisting Escherichia coli tRNA/synthetase pair was attempted. The methodinvolves eliminating the tRNA's affinity toward its cognate synthetaseby mutating nucleotides at the tRNA-synthetase interface whilepreserving its orthogonality to other synthetases and its ability tofunction in translation. Using the cognate wild-type synthetase as thestarting template, a mutant synthetase is then evolved that uniquelyrecognizes the engineered orthogonal tRNA. Based on an analysis of theX-ray crystal structure of Escherichia coli glutaminyl-tRNA synthetase(GlnRS) complexed with tRNAGln2, three sites (“knobs”) in tRNAGln2 wereidentified which make specific contacts with GlnRS. See, e.g., D. R.Liu, T. J. Magliery and P. G. Schultz, Chem. Biol., 4:685 (1997); and,D. R. Liu, T. J. Magliery, M. Pastrnak and P. G. Schultz, Proc. Natl.Acad. Sci. USA, 94:10092 (1997). These sites were mutated in the tRNA,and mutant suppressor tRNAs containing all possible combinations ofknobs 1, 2, and 3 were generated and tested individually by in vitroaminoacylation with GlnRS and in vitro suppression of amber mutants ofchorismate mutase. A mutant tRNA (O-tRNA) bearing all three-knobmutations was shown to be orthogonal to all endogenous Escherichia colisynthetases and competent in translation. Next, multiple rounds of DNAshuffling together with oligonucleotide-directed mutagenesis were usedto generate libraries of mutant GlnRS's. These mutant enzymes wereselected for their ability to acylate the O-tRNA in vivo usingEscherichia coli strain BT235. Only if a mutant GlnRS charges the O-tRNAwith glutamine can the genomic amber codon in lacZ be suppressed,enabling BT235 cells to grow on lactose minimal media. Several mutantsynthetases surviving each round of selection were purified and assayedin vitro. The ratio of wild-type (wt) tRNAGln acylation to O-tRNAacylation by mutant synthetase decreased significantly upon multiplerounds of selection. However, no mutant Escherichia coli GlnRS's havebeen evolved that charge the O-tRNA more efficiently than wild-typeEscherichia coli tRNAGln2. The best mutant evolved after seven rounds ofDNA shuffling and selection acylates the O-tRNA at only one-ninth therate of wt tRNAGln. However, these experiments failed to produce asynthetase candidate with the desired properties, e.g., a synthetasethat does not acylate any wt tRNA, since misacylation of a wt tRNA withan unnatural amino acid could result in a lethal phenotype. In addition,the mutations within the tRNA interact in complicated, non-additive wayswith respect to both aminoacylation and translation. See, D. R. Liu, T.J. Magliery and P. G. Schultz, Chem. Biol., 14:685 (1997). Thus,alternative methods are typically used to provide a functional pair withthe desired properties.

A second strategy for generating an orthogonal tRNA/synthetase pairinvolves importing a tRNA/synthetase pair from another organism intoEscherichia coli. The properties of the heterologous synthetasecandidate include, e.g., that it does not charge any Escherichia colitRNA, and the properties of the heterologous tRNA candidate include,e.g., that it is not acylated by any Escherichia coli synthetase. Inaddition, the suppressor tRNA derived from the heterologous tRNA isorthogonal to all Escherichia coli synthetases. Schimmel et al. reportedthat Escherichia coli GlnRS (EcGlnRS) does not acylate Saccharomycescerevisiae tRNAGln(EcGlnRS lacks an N-terminal RNA-binding domainpossessed by Saccharomyces cerevisiae GlnRS (ScGlnRS)). See, E. F.Whelihan and P. Schimmel, EMBO J., 16:2968 (1997). The Saccharomycescerevisiae amber suppressor tRNAGln(SctRNAGlnCUA) was then analyzed todetermine whether it is also not a substrate for EcGlnRS. In vitroaminoacylation assays showed this to be the case; and in vitrosuppression studies show that the SctRNAGlnCUA is competent intranslation. See, e.g., D. R. Liu and P. G. Schultz, Proc. Natl. Acad.Sci. USA, 96:4780 (1999). It was further shown that ScGlnRS does notacylate any Escherichia coli tRNA, only the SctRNAGlnCUA in vitro. Thedegree to which ScGlnRS is able to aminoacylate the SctRNAGlnCUA inEscherichia coli was also evaluated using an in vivo complementationassay. An amber nonsense mutation was introduced at a permissive site inthe β-lactamase gene. Suppression of the mutation by an amber suppressortRNA should produce full-length β-lactamase and confer ampicillinresistance to the cell. When only SctRNAGlnCUA is expressed, cellsexhibit an IC₅₀ of 20 μg/mL ampicillin, indicating virtually noacylation by endogenous Escherichia coli synthetases; when SctRNAGlnCUAis coexpressed with ScGlnRS, cells acquire an IC₅₀ of about 500 μg/mLampicillin, demonstrating that ScGlnRS acylates SctRNAGlnCUA efficientlyin Escherichia coli. See, D. R. Liu and P. G. Schultz, Proc. Natl. Acad.Sci. USA, 96:4780 (1999). The Saccharomyces cerevisiae tRNAGInCUA/GlnRSis orthogonal to Escherichia coli.

This strategy was later applied to a tRNA^(Asp)/AspRS system.

Saccharomyces cerevisiae tRNA^(Asp) is known to be orthogonal toEscherichia coli synthetases. See, e.g., B. P. Doctor and J. A. Mudd, J.Biol. Chem., 238:3677 (1963); and, Y. Kwok and J. T. Wong, Can. J.Biochem., 58:213 (1980). It was demonstrated that an amber suppressortRNA derived from it (SctRNAAspCUA) is also orthogonal in Escherichiacoli using the in vivo β-lactamase assay described above. However, theanticodon of tRNA^(Asp) is a critical recognition element of AspRS, see,e.g., R. Giege, C. Florentz, D. Kern, J. Gangloff, G. Eriani and D.Moras, Biochimie, 78:605 (1996), and mutation of the anticodon to CUAresults in a loss of affinity of the suppressor for AspRS. AnEscherichia coli AspRS E93K mutant has been shown to recognizeEscherichia coli amber suppressor tRNAAspCUA about an order of magnitudebetter than wt AspRS. See, e.g., F. Martin, ‘Thesis’, Universite LouisPasteur, Strasbourg, France, 1995. It was speculated that introductionof the related mutation in Saccharomyces cerevisiae AspRS (E188K) mightrestore its affinity for SctRNAAspCUA. It was determined that theSaccharomyces cerevisiae AspRS(E188K) mutant does not acylateEscherichia coli tRNAs, but charges SctRNAAspCUA with moderateefficiency as shown by in vitro aminoacylation experiments. See, e.g.,M. Pastrnak, T. J. Magliery and P. G. Schultz, Helv. Chim. Acta, 83:2277(2000). Although the SctRNAAspCUA/ScAspRS(E188K) can serve as anotherorthogonal pair in Escherichia coli, it possesses weak activity.

A similar approach involves the use of a heterologous synthetase as theorthogonal synthetase but a mutant initiator tRNA of the same organismor a related organism as the orthogonal tRNA. RajBhandary and coworkersfound that an amber mutant of human initiator tRNAfMet is acylated byEscherichia coli GlnRS and acts as an amber suppressor in yeast cellsonly when EcGlnRS is coexpressed. See, A. K. Kowal, C. Kohrer and U. L.RajBhandary, Proc. Natl. Acad. Sci. USA, 98:2268 (2001). This pair thusrepresents an orthogonal pair for use in yeast. Also, an Escherichiacoli initiator tRNAfMet amber mutant was found that is inactive towardany Escherichia coli synthetases. A mutant yeast TyrRS was selected thatcharges this mutant tRNA, resulting in an orthogonal pair in Escherichiacoli. See, A. K. Kowal, et al, (2001), supra.

The prokaryotic and eukaryotic tRNATyr/TyrRS pairs have significantdifferences: the identity elements of prokaryotic tRNATyr include a longvariable arm in contrast to the short arm of eukaryotic tRNATyr. Inaddition, eukaryotic tRNATyr contains a C1:G72 positive recognitionelement whereas prokaryotic tRNATyr has no such consensus base pair. Invitro studies have also shown that tRNATyr of Saccharomyces cerevisiaeand H. sapiens cannot be aminoacylated by bacterial synthetases, nor dotheir TyrRS aminoacylate bacterial tRNA. See, e.g., K. Wakasugi, C. L.Quinn, N. Tao and P. Schimmel, EMBO J., 17:297 (1998); and, T. A.Kleeman, D. Wei, K. L. Simpson and E. A. First, J. Biol. Chem.,272:14420 (1997). But, in spite of all these promising features fororthogonality, in vivo β-lactamase complementation assays showed thatthe amber suppressor tRNATyrCUA derived from both Saccharomycescerevisiae and H sapiens are not orthogonal in Escherichia coli. See,e.g., L. Wang, T. J. Magliery, D. R. Liu and P. G. Schultz, J. Am. Chem.Soc., 122:5010 (2000). The susceptibility of the suppressor tRNA toacylation by Escherichia coli synthetases is due to the change of onesingle nucleotide in the anticodon (G34 to C34).

Using the methods of the present invention, the pairs and components ofpairs desired above are evolved to generate orthogonal tRNA/synthetasepairs that possess desired characteristic, e.g., that can preferentiallyaminoacylate an O-tRNA with an unnatural amino acid.

Source and Host Organisms

The orthogonal tRNA-RS pair, e.g., derived from at least a firstorganism or at least two organisms, which can be the same or different,can be used in a variety of organisms, e.g., a second organism. Thefirst and the second organisms of the methods of the present inventioncan be the same or different. In one embodiment, the first organism is aprokaryotic organism, e.g., Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus,Halobacterium, P. furiosus, P. horikoshii, A. pernix, T. thermophilus,or the like. Alternatively, the first organism is a eukaryotic organism,e.g., plants (e.g., complex plants such as monocots, or dicots), algae,protists, fungi (e.g., yeast, etc), animals (e.g., mammals, insects,arthropods, etc.), or the like. In another embodiment, the secondorganism is a prokaryotic organism, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, e.g., plants, fungi, animals, or the like.

As described above, the individual components of a pair can be derivedfrom the same organism or different organisms. For example, tRNA can bederived from a prokaryotic organism, e.g., an archaebacterium, such asMethanococcus jannaschii and Halobacterium NRC-1 or a eubacterium, suchas Escherichia coli, while the synthetase can be derived from same oranother prokaryotic organism, such as, Methanococcus jannaschii,Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, P.furiosus, P. horikoshii, A. pernix, T. thermophilus, Halobacterium,Escherichia coli or the like.

Eukaryotic sources can also be used, e.g., plants (e.g., complex plantssuch as monocots, or dicots), algae, protists, fungi (e.g., yeast,etc.), animals (e.g., mammals, insects, arthropods, etc.), or the like.

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set fromthe second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set, where the first set andthe duplicate cell set are grown in the presence of a selection agent,and where the surviving cells comprise the orthogonal tRNA-tRNAsynthetase pair for use in the in the in vivo translation system of thesecond organism. In one embodiment, comparing and selecting includes anin vivo complementation assay. In another embodiment, the concentrationof the selection agent is varied.

For example, a tRNA/synthetase pair can be chosen based on where theidentity elements, which are recognition sites of the tRNA for thesynthetase, are found. For example, a tRNA/synthetase pair is chosenwhen the identity elements are outside of the anticodon, e.g., thetRNATyr/TyrRS pair from the archaebacterial Methanococcus jannaschii.This TyrRS is missing most of the non-conserved domain binding for theanticodon loop of its tRNATyr, but can discriminate tRNA with C1:G72from that with G1:C72. Furthermore, the Methanococcus jannaschii TyrRS(MjTyrRS) aminoacylates Saccharomyces cerevisiae but not Escherichiacoli crude tRNA. See, e.g., B. A. Steer and P. Schimmel, J. Biol. Chem.,274:35601 (1999). Using an in vivo complementation assay with anexpression vector containing a reporter gene, e.g., β-lactamase gene,with at least one selector codon, cells expressing the Methanococcusjannaschii tRNATyrCUA (Mj tRNATyrCUA) alone survive to an IC₅₀ of 55μg/mL ampicillin; cells coexpressing Mj tRNATyrCUA with its TyrRSsurvive to an IC₅₀ of 1220 ug/mL ampicillin. Although Mj tRNATyrCUA isless orthogonal in Escherichia coli than the SctRNAGlnCUA (IC₅₀ 20μg/mL), the MjTyrRS has higher aminoacylation activity toward itscognate amber suppressor tRNA. See, e.g., L. Wang, T. J. Magliery, D. R.Liu and P. G. Schultz, J. Am. Chem. Soc., 122:5010 (2000). As a result,Methanococcus jannaschii/TyrRS is identified as an orthogonal pair inEscherichia coli and can be selected for use in an in vivo translationsystem.

Unnatural Amino Acids

A wide variety of unnatural amino acids can be used in the methods ofthe invention. The unnatural amino acid can be chosen based on desiredcharacteristics of the unnatural amino acid, e.g., function of theunnatural amino acid, such as modifying protein biological propertiessuch as toxicity, biodistribution, or half life, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic properties, ability to react with other molecules (eithercovalently or noncovalently), or the like.

As used herein an “unnatural amino acid” refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand the following twenty genetically encoded alpha-amino acids: alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.The generic structure of an alpha-amino acid is illustrated by FormulaI:

An unnatural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See, e.g., any biochemistry text such asBiochemistry by L. Stryer, 3^(rd) ed. 1988, Freeman and Company, NewYork, for structures of the twenty natural amino acids. Note that, theunnatural amino acids of the present invention may be naturallyoccurring compounds other than the twenty alpha-amino acids above.Because the unnatural amino acids of the invention typically differ fromthe natural amino acids in side chain only, the unnatural amino acidsform amide bonds with other amino acids, e.g., natural or unnatural, inthe same manner in which they are formed in naturally occurringproteins. However, the unnatural amino acids have side chain groups thatdistinguish them from the natural amino acids. For example, R in FormulaI optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-,hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether,thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid,hydroxylamine, amino group, or the like or any combination thereof.Other unnatural amino acids of interest include, but are not limited to,amino acids comprising a photoactivatable cross-linker, spin-labeledamino acids, fluorescent amino acids, metal binding amino acids,metal-containing amino acids, radioactive amino acids, amino acids withnovel functional groups, amino acids that covalently or noncovalentlyinteract with other molecules, photocaged and/or photoisomerizable aminoacids, amino acids comprising biotin or a biotin analogue, glycosylatedamino acids such as a sugar substituted serine, other carbohydratemodified amino acids, keto containing amino acids, amino acidscomprising polyethylene glycol or polyether, heavy atom substitutedamino acids, chemically cleavable and/or photocleavable amino acids,amino acids with an elongated side chains as compared to natural aminoacids, e.g., polyethers or long chain hydrocarbons, e.g., greater thanabout 5 or greater than about 10 carbons, carbon-linked sugar-containingamino acids, redox-active amino acids, amino thioacid containing aminoacids, and amino acids comprising one or more toxic moiety.

In addition to unnatural amino acids that contain novel side chains,unnatural amino acids also optionally comprise modified backbonestructures, e.g., as illustrated by the structures of Formula II andIII:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which may be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids α-aminothiocarboxylates, e.g.,with side chains corresponding to the common twenty natural amino acidsor unnatural side chains. In addition, substitutions at the α-carbonoptionally include L, D, or α-α-disubstituted amino acids such asD-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and thelike. Other structural alternatives include cyclic amino acids, such asproline analogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, ∃ and γ amino acids such as substituted ∃-alanine and γ-aminobutyric acid.

For example, many unnatural amino acids are based on natural aminoacids, such as tyrosine, glutamine, phenylalanine, and the like.Tyrosine analogs include para-substituted tyrosines, ortho-substitutedtyrosines, and meta substituted tyrosines, wherein the substitutedtyrosine comprises an acetyl group, a benzoyl group, an amino group, ahydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropylgroup, a methyl group, a C₆-C₂₀ straight chain or branched hydrocarbon,a saturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, or the like. In addition, multiply substitutedaryl rings are also contemplated. Glutamine analogs of the inventioninclude, but are not limited to, α-hydroxy derivatives, γ-substitutedderivatives, cyclic derivatives, and amide substituted glutaminederivatives. Example phenylalanine analogs include, but are not limitedto, meta-substituted phenylalanines, wherein the substituent comprises ahydroxy group, a methoxy group, a methyl group, an allyl group, anacetyl group, or the like. Specific examples of unnatural amino acidsinclude, but are not limited to, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine, and the like. The structures of a variety ofnon-limiting unnatural amino acids are provided in the figures, e.g.,FIGS. 29, 30, and 31.

Typically, the unnatural amino acids of the invention are selected ordesigned to provide additional characteristics unavailable in the twentynatural amino acids. For example, unnatural amino acid are optionallydesigned or selected to modify the biological properties of a protein,e.g., into which they are incorporated. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, solubility, stability,e.g., thermal, hydrolytic, oxidative, resistance to enzymaticdegradation, and the like, facility of purification and processing,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic activity, redox potential,half-life, ability to react with other molecules, e.g., covalently ornoncovalently, and the like.

Further details regarding unnatural amino acids are described incorresponding application, “In vivo Incorporation of Unnatural AminoAcids”, attorney docket number 54-000120PC/US, filed Apr. 19, 2002 (U.S.Ser. No. 10/126,927, now U.S. Pat. No. 7,045,337), which is incorporatedherein by reference.

Use of Mutant tRNA and O-RS and O-tRNA/O-RS Pairs

The compositions of the present invention and compositions made by themethods of the present invention optionally are in a cell. TheO-tRNA/O-RS pairs or individual components of the present invention canthen be used in a host system's translation machinery, which results inan unnatural amino acid being incorporated into a protein. Thecorresponding patent application “In vivo Incorporation of UnnaturalAmino Acids”, attorney docket number 54-000120PC/US by Schultz, et al.(U.S. Ser. No. 10/126,927, now U.S. Pat. No. 7,045,337) describes thisprocess and is incorporated herein by reference. For example, when anO-tRNA/O-RS pair is introduced into a host, e.g., Escherichia coli, thepair leads to the in vivo incorporation of an unnatural amino acid,e.g., a synthetic amino acid, such as O-methyl-L-tyrosine, which can beexogenously added to the growth medium, into a protein, e.g.,dihydrofolate reductase or a therapeutic protein such as EPO, inresponse to a selector codon, e.g., an amber nonsense codon. Optionally,the compositions of the present invention can be in an in vitrotranslation system, or in an in vivo system(s).

Nucleic Acid and Polypeptide Sequence Variants

As described above and below, the invention provides for nucleic acidpolynucleotide sequences and polypeptide amino acid sequences, e.g.,O-tRNAs and O-RSs, and, e.g., compositions and methods comprising saidsequences. Examples of said sequences, e.g., O-tRNAs and O-RSs aredisclosed herein. However, one of skill in the art will appreciate thatthe invention is not limited to those sequences disclosed herein. One ofskill will appreciate that the present invention also provides manyrelated and unrelated sequences with the functions described herein,e.g., encoding an O-tRNA or an O-RS.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences, wherein the variants hybridize toat least one disclosed sequence, are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques, are alsoincluded in the invention.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties, are also readily identified as being highlysimilar to a disclosed construct. Such conservative variations of eachdisclosed sequence are a feature of the present invention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences, see, Table 1below. One of skill will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 5%, moretypically less than 4%, 2% or 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thedeletion of an amino acid, addition of an amino acid, or substitution ofan amino acid with a chemically similar amino acid. Thus, “conservativevariations” of a listed polypeptide sequence of the present inventioninclude substitutions of a small percentage, typically less than 5%,more typically less than 2% or 1%, of the amino acids of the polypeptidesequence, with a conservatively selected amino acid of the sameconservative substitution group. Finally, the addition of sequenceswhich do not alter the encoded activity of a nucleic acid molecule, suchas the addition of a non-functional sequence, is a conservativevariation of the basic nucleic acid.

TABLE 1 Conservative Substitution Groups 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Trytophan (W)

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of theinvention, including conservative variations of nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. In addition,target nucleic acids which hybridize to the nucleic acids represented bySEQ ID NO:1-3 or SEQ ID NO:4-34 (see, Table 5) under high, ultra-highand ultra-ultra high stringency conditions are a feature of theinvention. Examples of such nucleic acids include those with one or afew silent or conservative nucleic acid substitutions as compared to agiven nucleic acid sequence.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at lest ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, N.Y.), as well asin Ausubel, infra. Hames and Higgins (1995) Gene Probes 1 IRL Press atOxford University Press, Oxford, England, (Hames and Higgins 1) andflames and Higgins (1995) Gene Probes 2 IRL Press at Oxford UniversityPress, Oxford, England (Hames and Higgins 2) provide details on thesynthesis, labeling, detection and quantification of DNA and RNA,including oligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, Sambrook, infra for a description of SSCbuffer). Often the high stringency wash is preceded by a low stringencywash to remove background probe signal. An example low stringency washis 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 5× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determininghighly stringent hybridization and wash conditions, the hybridizationand wash conditions are gradually increased (e.g., by increasingtemperature, decreasing salt concentration, increasing detergentconcentration and/or increasing the concentration of organic solventssuch as formalin in the hybridization or wash), until a selected set ofcriteria are met. For example, the hybridization and wash conditions aregradually increased until a probe binds to a perfectly matchedcomplementary target with a signal to noise ratio that is at least 5× ashigh as that observed for hybridization of the probe to an unmatchedtarget.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids. A target nucleic acid which hybridizes to a probe undersuch conditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids. A target nucleic acid which hybridizesto a probe under such conditions, with a signal to noise ratio of atleast ½ that of the perfectly matched complementary target nucleic acidis said to bind to the probe under ultra-ultra-high stringencyconditions.

Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Unique Subsequences

In one aspect, the invention provides a nucleic acid which comprises aunique subsequence in a nucleic acid selected from the sequences ofO-tRNAs and O-RSs disclosed herein, e.g., SEQ ID NO:1-3 or SEQ IDNO:4-34 (see, Table 5). The unique subsequence is unique as compared toa nucleic acid corresponding to any previously known O-tRNA or O-RSnucleic acid sequence, e.g., as found in Genbank. Alignment can beperformed using, e.g., BLAST set to default parameters. Any uniquesubsequence is useful, e.g., as a probe to identify the nucleic acids ofthe invention.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from the sequences of O-RSsdisclosed herein, e.g., SEQ ID NO:35-66 (see, Table 5). Here, the uniquesubsequence is unique as compared to a polypeptide corresponding to anyof known polypeptide sequence.

The invention also provides for target nucleic acids which hybridizesunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from thesequences of O-RSs wherein the unique subsequence is unique as comparedto a polypeptide corresponding to any of the control polypeptides.Unique sequences are determined as noted above.

Sequence Comparison, Identity, and Homology

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding an O-tRNA or O-RS, or theamino acid sequence of an O-RS) refers to two or more sequences orsubsequences that have at least about 60%, preferably 80%, mostpreferably 90-95% nucleotide or amino acid residue identity, whencompared and aligned for maximum correspondence, as measured using asequence comparison algorithm or by visual inspection. Such“substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably,“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see, Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the invention provide a variety of newpolypeptide sequences (e.g., comprising unnatural amino acids in thecase of proteins synthesized in the translation systems herein, or,e.g., in the case of the novel synthetases herein, novel sequences ofstandard amino acids), the polypeptides also provide new structuralfeatures which can be recognized, e.g., in immunological assays. Thegeneration of antisera which specifically bind the polypeptides of theinvention, as well as the polypeptides which are bound by such antisera,are a feature of the invention.

For example, the invention includes synthetase proteins thatspecifically bind to or that are specifically immunoreactive with anantibody or antisera generated against an immunogen comprising an aminoacid sequence selected from one or more SEQ ID NO:35-66 (see, Table 5).To eliminate cross-reactivity with other homologues, the antibody orantisera is subtracted with available synthetases, such as the wild-typeMethanococcus jannaschii (M. jannaschii) tyrosyl synthetase (TyrRS),e.g., the “control” polypeptides. Where the wild-type Methanococcusjannaschii (M. jannaschii) tyrosyl synthetase (TyrRS) corresponds to anucleic acid, a polypeptide encoded by the nucleic acid is generated andused for antibody/antisera subtraction purposes.

In one typical format, the immunoassay uses a polyclonal antiserum whichwas raised against one or more polypeptide comprising one or more of thesequences corresponding to one or more of SEQ ID NO:35-66 (see, Table 5)or a substantial subsequence thereof (i.e., at least about 30% of thefull length sequence provided). The set of potential polypeptideimmunogens derived from SEQ ID NO:35-66 (see, Table 5) are collectivelyreferred to below as “the immunogenic polypeptides.” The resultingantisera is optionally selected to have low cross-reactivity against thecontrol synthetase homologues and any such cross-reactivity is removed,e.g., by immunoabsorbtion, with one or more of the control synthetasehomologues, prior to use of the polyclonal antiserum in the immunoassay.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein can be produced in arecombinant cell. An inbred strain of mice (used in this assay becauseresults are more reproducible due to the virtual genetic identity of themice) is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity.Additional references and discussion of antibodies is also found hereinand can be applied here to defining polypeptides by immunoreactivity).Alternatively, one or more synthetic or recombinant polypeptide derivedfrom the sequences disclosed herein is conjugated to a carrier proteinand used as an immunogen.

Polyclonal sera are collected and titered against the immunogenicpolypeptide in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control synthetase polypeptidesto produce subtracted pooled titered polyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for crossreactivity against the control homologues in a comparative immunoassay.In this comparative assay, discriminatory binding conditions aredetermined for the subtracted titered polyclonal antisera which resultin at least about a 5-10 fold higher signal to noise ratio for bindingof the titered polyclonal antisera to the immunogenic synthetase ascompared to binding to the control synthetase homologues. That is, thestringency of the binding reaction is adjusted by the addition ofnon-specific competitors such as albumin or non-fat dry milk, and/or byadjusting salt conditions, temperature, and/or the like. These bindingconditions are used in subsequent assays for determining whether a testpolypeptide (a polypeptide being compared to the immunogenicpolypeptides and/or the control polypeptides) is specifically bound bythe pooled subtracted polyclonal antisera. In particular, testpolypeptides which show at least a 2-5× higher signal to noise ratiothan the control synthetase homologues under discriminatory bindingconditions, and at least about a V2 signal to noise ratio as compared tothe immunogenic polypeptide(s), shares substantial structural similaritywith the immunogenic polypeptide as compared to known synthetases, andis, therefore a polypeptide of the invention.

In another example, immunoassays in the competitive binding format areused for detection of a test polypeptide. For example, as noted,cross-reacting antibodies are removed from the pooled antisera mixtureby immunoabsorbtion with the control polypeptides. The immunogenicpolypeptide(s) are then immobilized to a solid support which is exposedto the subtracted pooled antisera. Test proteins are added to the assayto compete for binding to the pooled subtracted antisera. The ability ofthe test protein(s) to compete for binding to the pooled subtractedantisera as compared to the immobilized protein(s) is compared to theability of the immunogenic polypeptide(s) added to the assay to competefor binding (the immunogenic polypeptides compete effectively with theimmobilized immunogenic polypeptides for binding to the pooledantisera). The percent cross-reactivity for the test proteins iscalculated, using standard calculations.

In a parallel assay, the ability of the control proteins to compete forbinding to the pooled subtracted antisera is optionally determined ascompared to the ability of the immunogenic polypeptide(s) to compete forbinding to the antisera. Again, the percent cross-reactivity for thecontrol polypeptides is calculated, using standard calculations. Wherethe percent cross-reactivity is at least 5-10× as high for the testpolypeptides as compared to the control polypeptides and or where thebinding of the test polypeptides is approximately in the range of thebinding of the immunogenic polypeptides, the test polypeptides are saidto specifically bind the pooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in acompetitive binding immunoassay as described herein to compare any testpolypeptide to the immunogenic and/or control polypeptide(s). In orderto make this comparison, the immunogenic, test and control polypeptidesare each assayed at a wide range of concentrations and the amount ofeach polypeptide required to inhibit 50% of the binding of thesubtracted antisera to, e.g., an immobilized control, test orimmunogenic protein is determined using standard techniques. If theamount of the test polypeptide required for binding in the competitiveassay is less than twice the amount of the immunogenic polypeptide thatis required, then the test polypeptide is said to specifically bind toan antibody generated to the immunogenic protein, provided the amount isat least about 5-10× as high as for the control polypeptide.

As an additional determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptides) until little or no binding of theresulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunosorbtion is detectable.This fully immunosorbed antisera is then tested for reactivity with thetest polypeptide. If little or no reactivity is observed (i.e., no morethan 2× the signal to noise ratio observed for binding of the fullyimmunosorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic protein.

General Techniques

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, cellculture and the like, include Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—ALaboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (supplemented through 2002) (“Ausubel”)). These textsdescribe mutagenesis, the use of vectors, promoters and many otherrelevant topics related to, e.g., the generation of orthogonal tRNA,orthogonal synthetases, and pairs thereof.

Various types of mutagenesis are used in the present invention, e.g., toproduce novel synthetases or tRNAs. They include but are not limited tosite-directed, random point mutagenesis, homologous recombination (DNAshuffling), mutagenesis using uracil containing templates,oligonucleotide-directed mutagenesis, phosphorothioate-modified DNAmutagenesis, mutagenesis using gapped duplex DNA or the like. Additionalsuitable methods include point mismatch repair, mutagenesis usingrepair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,e.g., involving chimeric constructs, are also included in the presentinvention. In one embodiment, mutagenesis can be guided by knowninformation of the naturally occurring molecule or altered or mutatednaturally occurring molecule, e.g., sequence, sequence comparisons,physical properties, crystal structure or the like.

The above texts and examples found herein describe these procedures aswell as the following publications and references cited within: Sieber,et al., Nature Biotechnology, 19:456-460 (2001); Ling et al., Approachesto DNA mutagenesis: an overview, Anal Biochem. 254(2): 157-178 (1997);Dale et al., Oligonucleotide-directed random mutagenesis using thephosphorothioate method, Methods Mol. Biol. 57:369-374 (1996); I. A.Lorimer, I. Pastan, Nucleic Acids Res. 23, 3067-8 (1995); W. P. C.Stemmer, Nature 370, 389-91 (1994); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Bass et al., Mutant Trp repressors with new DNA-binding specificities,Science 242:240-245 (1988); Fritz et al., Oligonucleotide-directedconstruction of mutations: a gapped duplex DNA procedure withoutenzymatic reactions in vitro, Nucl. Acids Res. 16: 6987-6999 (1988);Kramer et al., Improved enzymatic in vitro reactions in the gappedduplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Sakamar and Khorana, Totalsynthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin), Nucl. AcidsRes. 14: 6361-6372 (1988); Sayers et al., Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Carter, Improved oligonucleotide-directed mutagenesisusing M13 vectors, Methods in Enzymol, 154: 382-403 (1987); Kramer &Fritz Oligonucleotide-directed construction of mutations via gappedduplex DNA, Methods in Enzymol. 154:350-367 (1987); Kunkel, Theefficiency of oligonucleotide directed mutagenesis, in Nucleic Acids &Molecular Biology (Eckstein, F. and Lilley, D. M. J. eds., SpringerVerlag, Berlin)) (1987); Kunkel et al., Rapid and efficientsite-specific mutagenesis without phenotypic selection, Methods inEnzymol. 154, 367-382 (1987); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987);Carter, Site-directed mutagenesis, Biochem. J. 237:1-7 (1986);Eghtedarzadeh & Henikoff, Use of oligonucleotides to generate largedeletions, Nucl. Acids Res. 14: 5115 (1986); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Wells et al., Importance of hydrogen-bondformation in stabilizing the transition state of subtilisin, Phil.Trans. R. Soc. Lond. A 317: 415-423 (1986); Botstein & Shortie,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter et al., Improved oligonucleotidesite-directed mutagenesis using M13 vectors, Nucl. Acids Res. 13:4431-4443 (1985); Grundstrom et al., Oligonucleotide-directedmutagenesis by microscale ‘shot-gun’ gene synthesis, Nucl. Acids Res.13: 3305-3316 (1985); Kunkel, Rapid and efficient site-specificmutagenesis without phenotypic selection, Proc. Natl. Acad. Sci. USA82:488-492 (1985); Smith, In vitro mutagenesis, Ann. Rev. Genet.19:423-462 (1985); Taylor et al., The use of phosphorothioate-modifiedDNA in restriction enzyme reactions to prepare nicked DNA, Nucl. AcidsRes. 13: 8749-8764 (1985); Taylor et al., The rapid generation ofoligonucleotide-directed mutations at high frequency usingphosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8787 (1985);Wells et al., Cassette mutagenesis: an efficient method for generationof multiple mutations at defined sites, Gene 34:315-323 (1985); Krameret al., The gapped duplex DNA approach to oligonucleotide-directedmutation construction, Nucl. Acids Res. 12: 9441-9456 (1984); Kramer etal., Point Mismatch Repair, Cell 38:879-887 (1984); Nambiar et al.,Total synthesis and cloning of a gene coding for the ribonuclease Sprotein, Science 223: 1299-1301 (1984); Zoller & Smith,Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors, Methods in Enzymol. 100:468-500 (1983); and Zoller & Smith,Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment, Nucleic Acids Res. 10:6487-6500 (1982). Additionaldetails on many of the above methods can be found in Methods inEnzymology Volume 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of synthetases, or altering tRNAs, aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes., 12:6159-6168 (1984).

In addition, essentially any nucleic acid can be custom or standardordered from any of a variety of commercial sources, such as The MidlandCertified Reagent Company, The Great American Gene Company, ExpressGenInc., Operon Technologies Inc. (Alameda, Calif.) and many others.

The present invention also relates to host cells and organisms for thein vivo incorporation of an unnatural amino acid via orthogonal tRNA/RSpairs. Host cells are genetically engineered (e.g., transformed,transduced or transfected) with the vectors of this invention, which canbe, for example, a cloning vector or an expression vector. The vectorcan be, for example, in the form of a plasmid, a bacterium, a virus, anaked polynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (From et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)). Berger, Sambrook, and Ausubel provide avariety of appropriate transformation methods.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms.

Other useful references, e.g. for cell isolation and culture (e.g., forsubsequent nucleic acid isolation) include Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg N.Y.) and Atlas and Parks (eds.) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intobacterial cells are available, any of which can be used in the presentinvention. These include: fusion of the recipient cells with bacterialprotoplasts containing the DNA, electroporation, projectile bombardment,and infection with viral vectors, etc. Bacterial cells can be used toamplify the number of plasmids containing DNA constructs of thisinvention. The bacteria are grown to log phase and the plasmids withinthe bacteria can be isolated by a variety of methods known in the art(see, for instance, Sambrook). In addition, a plethora of kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) andselection markers for both prokaryotic and eukaryotic systems. Vectorsare suitable for replication and integration in prokaryotes, eukaryotes,or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, etal., Nature, 328:731 (1987); Schneider, B., et al., Protein Expr. Purif.6435:10 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue ofBacteria and Bacteriophages useful for cloning is provided, e.g., by theATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992)Gherna et al. (eds.) published by the ATCC. Additional basic proceduresfor sequencing, cloning and other aspects of molecular biology andunderlying theoretical considerations are also found in Watson et al.(1992) Recombinant DNA Second Edition Scientific American Books, NY.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Improvement of Orthogonality of a tRNA from Methanococcusjannaschii

Because of the complex nature of tRNA-synthetase interactions that arerequired to achieve a high degree of fidelity in protein translation,the rational design of orthogonal tRNA-synthetase pairs is difficult.This example describes methods that exploit the poor cross recognitionof some interspecies tRNA-synthetase pairs, coupled with subsequent invivo evolution of tRNAs with enhanced orthogonality. See, also, L. Wangand P. G. Schultz, Chem. Biol., 8:883 (2001). Specifically, a library ofamber suppressor tRNAs derived from Methanococcus jannaschii tRNATyr wasgenerated. tRNATyrCUAs that are substrates for endogenous Escherichiacoli aminoacyl-tRNA synthetases were deleted from the pool by negativeselection based on suppression of amber nonsense mutations in thebarnase gene. The remaining tRNATyrCUAs were then selected for theirability to suppress amber nonsense codons in the ∃-lactamase gene in thepresence of the cognate Methanococcus jannaschii tyrosyl-tRNA synthetase(TyrRS). Four mutant suppressor tRNAs were selected that are poorersubstrates for Escherichia coli synthetases than Methanococcusjannaschii tRNATyrCUA, but still can be charged efficiently byMethanococcus jannaschii TyrRS. The mutant suppressor tRNATyrCUAtogether with the Methanococcus jannaschii TyrRS provide a usefulorthogonal tRNA-synthetase pair for the in vivo incorporation ofunnatural amino acids into proteins.

The tRNATyr of Methanococcus jannaschii, an archaebacterium, hasdifferent identity elements from those of Escherichia coli tRNATyr. Inparticular, the Escherichia coli tRNATyr has a G1C72 pair in theacceptor stem while the Methanococcus jannaschii tRNATyr has a C1G72pair. An amber suppressor tRNA derived from Methanococcus jannaschiitRNATyr was shown not to be efficiently aminoacylated by the Escherichiacoli synthetases, but functions efficiently in protein translation inEscherichia coli. See, e.g., L. Wang, T. J. Magliery, D. R. Liu, P. G.Schultz, A new functional suppressor tRNA/aminoacyl-tRNA synthetase pairfor the in vivo incorporation of unnatural amino acids into proteins, J.Am. Chem. Soc. 122:5010-5011 (2000). In addition, the Methanococcusjannaschii TyrRS, which has only a minimalist anticodon-loop-bindingdomain, does not aminoacylate Escherichia coli tRNAs, but stillefficiently aminoacylates its own suppressor tRNATyrCUA. See, e.g., B.A. Steer, P. Schimmel, Major anticodon-binding region missing from anarchaebacterial tRNA synthetase, J. Biol. Chem. 274 (1999) 35601-35606;and, Wang et al., (2000), supra.

To test the orthogonality of this suppressor tRNA in Escherichia coli,an amber codon was introduced at a permissive site (Ala184) in the∃-lactamase gene. See, e.g., D. R. Liu, P. G. Schultz, Progress towardthe evolution of an organism with an expanded genetic code, Proc. Natl.Acad. Sci. USA 96:4780-4785 (1999). Those tRNAs that can be charged byEscherichia coli synthetases will suppress the amber codon and allowcells to live in the presence of ampicillin. The Methanococcusjannaschii tRNATyrCUA suppresses the amber codon in the ∃-lactamase genewith an IC₅₀ value of 56 μg/ml ampicillin. See Wang et al., (2000),supra. In contrast, the orthogonal tRNAGlnCUA derived from Saccharomycescerevisiae tRNAGln2 has an IC₅₀ of 21 μg/ml ampicillin when tested inthe same assay. See Liu & Schultz, (1999), supra. The IC₅₀ forEscherichia coli in the absence of any suppressor tRNA is 10 μg/mlampicillin. This result shows that the Methanococcus jannaschiitRNATyrCUA is a better substrate for Escherichia coli synthetases thanthe tRNAGlnCUA. Consequently, if the Methanococcus jannaschii tRNATyrCUAis used in vivo to deliver unnatural amino acids into proteins inEscherichia coli, it can also be mischarged with natural amino acids byEscherichia coli synthetases, leading to heterogeneous amino acidincorporation.

The improvement of the orthogonality of the Methanococcus jannaschiitRNATyrCUA was accomplished by the introduction of ‘negative recognitiondeterminants’ to prevent recognition by endogenous Escherichia colisynthetases. These mutations should not strongly interfere with thetRNA's interaction with its cognate Methanococcus jannaschii TyrRS orthe ribosome. Since Methanococcus jannaschii TyrRS lacks most of theanticodon-binding domain, see, e.g., B. A. Steer, P. Schimmel, Majoranticodon-binding region missing from an archaebacterial tRNAsynthetase, J. Biol. Chem. 274:35601-35606 (1999), mutations introducedat the anticodon loop of the tRNA are expected to have a minimal effecton TyrRS recognition. An anticodon-loop library with four randomizednucleotides was constructed. See FIG. 9. Given the various combinationsand locations of identity elements for various Escherichia coli tRNAs,mutations at additional positions can increase the likelihood of findinga mutant tRNA with the desired properties. Thus, a second librarycontaining mutations at nonconserved positions in all of the tRNA loops(all-loop library) was also constructed. See FIG. 9. Conservednucleotides were not randomized so as to maintain the tertiaryinteractions that stabilize the ‘L’-shaped structure of the tRNA. See,e.g., G. Dirheimer, G. Keith, P. Dumas, E. Westhof, Primary, secondary,and tertiary structures of tRNAs, in: D. Söll, U. L. RajBhandary (eds.),tRNA Structure, Biosynthesis, and Function, ASM Press, Washington, D.C.,1995, pp. 93-126; and, R. Giegé, M. Sissler, C. Florentz, Universalrules and idiosyncratic features in tRNA identity, Nucleic Acids Res.26:5017-5035 (1998). Stem nucleotides were also not mutated sincesubstitution of one such nucleotide requires a compensatory mutation.The 11 nucleotides (C16, C17, U17a, U20, C32, G37, A38, U45, U47, A59,and U60) were randomized. See, FIG. 9. The theoretical size of thislibrary is about 4.19×10⁶, and a library with a size of about 1.93×10⁸colony-forming units was constructed to ensure complete coverage of themutant library.

The methods used an Escherichia coli strain, e.g., DH10B, which wasobtained from Gibco/BRL. Suppressor tRNA expression plasmids werederived from a plasmid, e.g., pAC123. See, e.g., D. R. Liu, T. J.Magliery, M. Pastrnak, P. G. Schultz, Engineering a tRNA andaminoacyl-tRNA synthetase for the site-specific incorporation ofunnatural amino acids into proteins in vivo, Proc. Natl. Acad. Sci. USA94:10091-10097 (1997). Plasmids for negative selections were derivedfrom plasmids, e.g., pBATS, pYsupA38B2 and pYsupA38B3 as describedbelow. See, e.g., K. Gabriel, W.H. McClain, A set of plasmidsconstitutively producing different RNA levels in Escherichia coli, J.Mol. Biol. 290 (1999) 385-389; and, Liu & Shultz, (1999), supra.

To select for a member of the Methanococcus jannaschii tRNA library withenhanced orthogonality, a combination of negative and positiveselections in the absence and presence of the cognate synthetase wasused. See FIG. 8. In the negative selection, selector codon(s), e.g.,amber nonsense, are introduced in a negative marker gene, e.g., a toxicgene, at e.g., a nonessential position. When a member of the mutated,e.g., suppressor, tRNA library is aminoacylated by endogenous (e.g.,Escherichia coli) synthetases (i.e. it is not orthogonal to theEscherichia coli synthetases), the selector codon is suppressed and thetoxic gene product produced leads to cell death. Only cells harboringorthogonal tRNAs or nonfunctional tRNAs can survive. All survivors arethen subjected to a positive selection in which a selector codon, e.g.,an amber codon, is placed in a positive selection marker, e.g., drugresistance gene at, e.g., a nonessential position. tRNAs are thenselected for their ability to be aminoacylated by the coexpressedcognate synthetase and to insert an amino acid in response to this ambercodon. Cells harboring nonfunctional tRNAs, or tRNAs that cannot berecognized by the synthetase of interest will be sensitive toantibiotic. Therefore, only tRNAs that (1) are not substrates forendogenous Escherichia coli synthetases; (2) can be aminoacylated by thesynthetase of interest; (3) are functional in translation will surviveboth selections.

A negative selection was chosen that takes advantage of the toxicity ofbarnase when produced in Escherichia coli in the absence of its naturalinhibitor barstar. See, e.g., R. W. Hartley, Barnase and barstar.Expression of its cloned inhibitor permits expression of a clonedribonuclease, J. Mol. Biol. 202:913-915 (1988). Amber codons wereintroduced at nonessential positions in the barnase gene based onanalysis of the three-dimensional structure of barnase. See, e.g., Liu &Schultz, (1999), supra. Because of barnase's extreme autotoxicity, a lowcopy number pSC101 origin was placed in the plasmid expressing barnase.In addition, different numbers of amber codons were tested to modulatethe stringency of the selection. Plasmid pSCB2 was used to express abarnase mutant with two amber stop codons at Gln2 and Asp44; plasmidpSCB3 contained an additional amber stop codon at Gly65.

For negative selection, a PCR fragment containing the β-lactamase geneand the pSC101 origin was generated from pBATS using the followingoligonucleotides: LW115, 5′-ATGCATGCTGCATTAATGAATCGGCCAACG-3′ (SEQ IDNO:67); LW116, 5″-TCCCCGCGGAGGTGGCACTTTTCGGGG-3′ (SEQ ID NO:68). DNAencoding barnase containing two (residues Gln2 and Asp44) or three(residues Gln2, Asp44 and Gly65) amber codons were obtained frompYsupA38B2 and pYsupA38B3, respectively, by digestion with SacII andSphI. Ligation of the above fragments afforded plasmids pSCB2 and pSCB3.The expression of barnase was under arabinose induction. Genes encodingdifferent suppressor tRNAs for in vivo expression were constructed fromtwo overlapping synthetic oligonucleotides (Operon, Alameda, Calif.,USA) by Klenow extension and inserted between the EcoRI and PstI sitesof pAC123 to generate pAC-YYG1 and pAC-JY, respectively, placingtranscription under control of the lpp promoter and the rrnC terminator.pAC-Cm is the control plasmid without any tRNA. To optimize the negativeselection conditions, competent DH10B cells harboring pSCB2 or pSCB3were transformed by electroporation with pAC-Cm, pAC-YYG1, and pAC-JY,separately. Single colonies were picked and grown in 2×YT withchloramphenicol (Cm, 34 μg/ml) and ampicillin (Amp, 100 μg/ml). Cellcultures grown overnight were washed twice with minimal media containing1% glycerol and 0.3 mM leucine (GMML), and resuspended in GMML with Cmand Amp to an OD600 of 0.1. After recovering at 30° C. for 10 min, intoone culture (set 1) was added 20 mM of arabinose to induce theexpression of barnase; no arabinose was added to the second culture (set2). At different time points, a small amount of cell culture was dilutedand plated on 2×YT agar with Cm and Amp to measure cell density. Fornegative selections of the suppressor tRNA libraries, the pAC plasmidscontaining the library were transformed into DH10B cells harboringpSCB2. Cells were quenched by addition of SOC medium and recovered at30° C. for 1 hour, then were washed with phosphate buffer and GMML, andcultured in 11 GMML. After recovering at 30° C. for 30 min, Cm, Amp, and20 mM arabinose were added. After 36 hours, cells were pelleted and pACplasmids were isolated and purified by agarose gel electrophoresis.

To optimize the selection conditions, two suppressor tRNAs were usedthat are known to be poorly recognized by the Escherichia colisynthetases. A mutant suppressor tRNATyr derived from Saccharomycescerevisiae (sc-tRNATyrCUA, expressed in pAC-YYG1) suppresses the ambercodon (Ala184TAG) in the ∃-lactamase gene, affording an IC₅₀ value of 12μg/ml ampicillin for Escherichia coli cells; and the suppressor tRNATyrderived from Methanococcus jannaschii (mj-tRNATyrCUA, expressed inpAC-JY) affords an IC₅₀ of 56 μg/ml ampicillin for host cells. See,e.g., Wang et al, (2000), supra. For comparison, the suppressortRNAGlnCUA derived from Saccharomyces cerevisiae tRNAGIn2 has an IC₅₀ of21 μg/ml ampicillin when tested in the same assay, and has beendemonstrated to be orthogonal to Escherichia coli synthetases in vitroand in vivo. See, e.g., Liu & Schultz, (1999), supra. Therefore, anegative selection that eliminates cells expressing mj-tRNATyrCUA, butallows the growth of cells expressing sc-tRNATyrCUA deletesnon-orthogonal suppressor tRNAs. Cells were grown in liquid minimalmedia containing 1% glycerol and 0.3 mM leucine (GMML) with appropriateantibiotics to maintain plasmid pSCB2 and the pAC plasmid. Arabinose wasadded to one set of cells (set 1) to induce the expression of thebarnase, while in set 2 no arabinose was added. The fraction of cellssurviving the selection was determined by the ratio of cell densities inset 1 relative to set 2. See FIG. 11: cells harboring the controlplasmid pAC-Cm (without suppressor tRNA) and plasmid pAC-YYG1 survived,while cells harboring plasmid pAC-JY largely died. When plasmid pSCB3was used, cells harboring plasmid pAC-JY started to grow in 24 hours.Therefore, the negative selection was carried out using pSCB2, whichencodes the barnase gene containing two amber codons under the aboveconditions for the library selection.

For positive selection, a plasmid, e.g., pBLAM-JYRS was constructed byinserting the Methanococcus jannaschii TyrRS gene from pBSA50 betweenNdeI and PstI sites of pBLAM-YQRS using oligonucleotides LW104,5′-GGAATTCCATTAGGACGAATTTGAAATG-3′ (SEQ ID NO:69); and LW105,5′-AAACTGCAGTTATAATCTCTTTCTAATTGGCTC-3′ (SEQ ID NO:70). See, e.g.,Steer, et al., (1999), supra; and, Liu & Schultz, (1999), supra. Tooptimize the positive selection conditions, competent DH10B cellsharboring pBLAM-JYRS were transformed with pAC-Cm, pAC-YYG1, and pAC-JY,separately. Single colonies were picked and grown in 2×YT with Cm andtetracycline (Tet, 40 μg/ml). In liquid selections, overnight cellcultures were diluted into 2×YT with Cm and Tet at a starting OD600 of0.1. Various concentrations of Amp were added, and cell growth wasmonitored by OD600. In plate selections, approximately 103 to 105 cellswere plated on two sets of 2×YT agar plates containing Cm and Tet, oneset of which contained 500 μg/ml Amp. For selections involving themutant tRNA library, pAC plasmids isolated from the cells from thenegative selection were transformed into competent DH10B cells harboringpBLAM-JYRS. Cells were recovered at 37° C. for 45 minutes, andapproximately 105 cells were plated onto each 2×YT agar plate containingCm, Tet and 500 μg/ml of Amp. After 24 hours, colonies were picked andre-grown in 6 ml 2×YT containing Cm, Tet and 200 μg/ml of Amp. DNA wasisolated and pAC plasmid was purified by agarose gel electrophoresis.

The positive selection is based on suppression of an amber stop codonintroduced at position Ala184 in the TEM-1 β-lactamase gene. PlasmidpBLAM-JYRS encodes the gene for the Methanococcus jannaschiityrosyl-tRNA synthetase and a ∃-lactamase with an amber mutation atAla184. pAC plasmids isolated from cells surviving the negativeselection were cotransformed with pBLAM-JYRS into Escherichia coli DH10Bcells. Cells harboring nonfunctional tRNAs or tRNAs that are poorsubstrates for the Methanococcus jannaschii synthetase die; those withtRNAs that can be charged by the synthetase survive. To test thefeasibility of the positive selection, two model suppressor tRNAs weretested in the presence of Methanococcus jannaschii TyrRS. Thesc-tRNATyrCUA has a G1:C72 base pair and is not charged efficiently byMethanococcus jannaschii TyrRS. When they were coexpressed in cells withthe Ala184amber β-lactamase mutant, cells survived to an IC₅₀ of 18μg/ml ampicillin. In contrast, cells containing the Methanococcusjannaschii tRNATyrCUA and the cognate TyrRS survive to an IC₅₀ of 1220μg/ml ampicillin. See, e.g., Wang, et al., (2000), supra. The modelpositive selection was first tried in liquid 2×YT medium. The growth ofcells harboring pBLAM-JYRS and different pAC plasmids in liquid 2×YTmedium with various concentrations of ampicillin are shown in FIG. 12,Panel A. Cells transformed with the mj-tRNATyrCUA grew at a faster rateand at higher concentrations of ampicillin. If cells were grown longerthan 24 hours, cells transformed with either pAC-Cm or pAC-YYG1 alsogrew to saturation. Therefore, the positive selection was carried out onplates with initial cell densities between 103 and 105 per plate. SeeFIG. 12, Panel B. The survival ratio (number of colonies on plates withampicillin relative to plates without ampicillin) did not changesignificantly with different initial cell densities, and was stable overthe growth time. The positive selection on ampicillin plates resulted inpreferential growth of cells with mj-tRNATyrCUA expressed. Therefore,for the library selection the positive selection was carried out onplates instead of in liquid medium.

The library of mutant tRNAs was generated by using the sequences of thetwo overlapping oligonucleotides used to construct the anticodon-looplibrary are (the tRNA sequence underlined): LW125, 5′-GGAATTC-3′; LW126,5′-AAAACTGCAG-3′ (SEQ ID NO: 71) (where N is equimolar of A, C, T or G).The sequences of oligonucleotides for the all-loop library are: LW145,5′-GGAATTC-3′ and LW146, 5′-AAAACTGCAG-3′ (SEQ ID NO:72). These geneswere inserted into pAC123 similarly as described above to afford thetRNA libraries.

The negative and positive selections were carried out in tandem asdescribed above on both the anticodon-loop and all-loop libraries. Theselected suppressor tRNAs were isolated and retransformed intoEscherichia coli DH10B harboring pBLAM to test the tRNA's orthogonalityto Escherichia coli synthetases. The tRNAs were then retransformed intoEscherichia coli harboring pBLAM-JYRS to test how efficiently the tRNAwas charged by Methanococcus jannaschii TyrRS. Sequencing of the clonesresulting from one round of negative and positive selection ofanticodon-loop library revealed that three independent tRNAs wereisolated. See FIG. 13. When cotransformed with pBLAM, all had lower IC₅₀values than the parent Methanococcus jannaschii tRNATyrCUA, indicatingthey are poorer substrates for Escherichia coli synthetases.

Mutant AA2 also had very high affinity for Methanococcus jannaschiiTyrRS. Although this mutant tRNA could be stably maintained inEscherichia coli, it slowed the growth rate of cells for unknownreasons. This effect likely led to the emergence of mutants AA3 and AA4,which both had a mutation outside of the randomization region. Cellsharboring AA3 or AA4 grew normally. Nevertheless, AA3 and AA4 wererelatively poor substrates for the Methanococcus jannaschii TyrRS.

Four independent tRNAs were selected from two rounds of negative andpositive selections using the all-loop library. See FIG. 13. All werepoorer substrates for the Escherichia coli synthetase than the parentMethanococcus jannaschii tRNATyrCUA, yet were still efficiently chargedby the Methanococcus jannaschii TyrRS as shown by the in vivoβ-lactamase assay. See Table 2. The IC₅₀ value for cells expressing thebest mutant, J17, was 12 μg/ml ampicillin, which is even lower than thatof cells with the orthogonal tRNAGlnCUA derived from Saccharomycescerevisiae expressed (21 μg/ml ampicillin). When J17 was coexpressedwith the Methanococcus jannaschii TyrRS, cells survived to an IC₅₀ valueof 436 μg/ml ampicillin, providing a selection window (ratio of IC₅₀value with TyrRS to IC50 value without TyrRS) of 35-fold. In addition,the expression of all these mutant tRNAs did not affect the growth ofEscherichia coli cells.

TABLE 2 In vivo β-lactamase assay of selected suppressor tRNAs IC₅₀(μg/ml of ampicillin) Coexpressed with Coexpressed with Suppressor tRNApBLAM pBLAM-JYRS mj-tRNATyrCUA 56 1220 No tRNATyrCUA 10 10 Mutant tRNAsselected from anticodon-loop library AA2 22 1420 AA3 10 110 AA4 12 135Mutant tRNAs selected from all-loop library Mutant tRNAs surviving bothselections J15 30 845 J17 12 436 J18 20 632 J22 14 459 Mutant tRNAssurviving negative selection only N11 11 16 N12 9 18 N13 10 12 N16 9 9

Plasmid pBLAM was used to express the β-lactamase gene with an ambercodon at Ala184; plasmid pBLAM-JYRS expressed the amber mutant and theTyrRS of Methanococcus jannaschii. Suppressor tRNAs were encoded on pACplasmid and cotransformed with pBLAM or pBLAM-JYRS in the assay.

To confirm the properties of the selected suppressor tRNAs, they weretested in another in vivo assay based on the suppression of an ambercodon in the chloramphenicol acetyltransferase (CAT) gene. In contrastto ∃-lactamase which is secreted into the periplasm, CAT localizes inthe cytoplasm. Moreover, ampicillin is bacteriocidal whilechloramphenicol is bacteriostatic. As shown in Table 3 below, theselected suppressor tRNAs also were orthogonal in the CAT assay,indicating their suitability for CAT selections.

TABLE 3 In vivo chloramphenicol acetyltransferase assay of selectedsuppressor tRNAs IC₅₀ (μg/ml of chloramphenicol) Suppressor tRNA pYConly pYC + pBK-JYRS mj-tRNATyrCUA 27 308 No tRNATyrCUA 3 3 J15 11 297J17 4 240 J18 6 284 J22 5 271pYC plasmids encoded the chloramphenicol acetyltransferase gene with anamber codon at Asp112 and different suppressor tRNAs listed in the leftcolumn of the table. pBK-JYRS was used to express the TyrRS ofMethanococcus jannaschii.

The in vivo complementation assay which is based on suppression of anamber codon in the ∃-lactamase gene was carried out as described. See,e.g., Liu & Schultz, (1999), supra; and, Wang, et al., (2000), supra. Inthe chloramphenicol acetyltransferase (CAT) assay, an amber codon wassubstituted for Asp112 in the CAT gene of pACYC184 to affordpACMD112TAG. See, e.g., M. Pastrnak, T. J. Magliery, P. G. Schultz, Anew orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair forevolving an organism with an expanded genetic code, Helv. Chim. Acta83:2277-2286 (2000). The genes encoding the suppressor tRNAs under thecontrol of the lpp promoter and rrnC terminator were excised from pACplasmids with NcoI and AvaI, and inserted into the pre-digestedpACMD112TAG to afford plasmids pYC-JY, pYC-J15, pYC-J17, pYC-J18, andpYC-J22, respectively. Plasmid pBK-JYRS, a derivative of pBR322, wasused to express the Methanococcus jannaschii TyrRS under the control ofthe Escherichia coli GlnRS promoter and terminator. The survival ofEscherichia coli DH10B cells transformed with pYC plasmid alone orcotransformed with pYC and pBK-JYRS was titrated against a wide range ofchloramphenicol concentrations added to the growth media, and IC50values were interpolated from the curves.

For comparison, four colonies were randomly picked that passed thenegative selection only, and tested the tRNAs using the in vivocomplementation assay. All of them had very low IC₅₀ values whentransformed with pBLAM, indicating the negative selection worked well.See Table 2. The IC₅₀ values were also low when cotransformed withpBLAM-JYRS, revealing that the positive selection functions to deletetRNAs that cannot be charged by the Methanococcus jannaschii TyrRS.

Analysis of the DNA sequences of the selected tRNAs yielded acharacteristic pattern of nucleotide substitutions. See FIG. 13. tRNAsthat passed both negative and positive selections all had C32 and T60unchanged, while G37 was mutated to A, and T17a was mutated to either Aor G. Some semi-conserved changes included mutation of A38 to either Cor A; mutation of T45 to either T or A; mutation of T47 to either G orT. Other mutations had no obvious common pattern. Twenty (20) tRNAs thatpassed the negative selection only were also sequenced, four of whichare shown in FIG. 13, and found they all lacked at least one of thecommon mutations listed above.

The preferred nucleotides in the selected mutant suppressor tRNAs canplay the following roles: (i) they can function as negative determinantsfor recognition by the Escherichia coli synthetases; (ii) they can beidentity elements for aminoacylation by Methanococcus jannaschii TyrRS;or (iii) they can also optimize the tRNA's interaction with Escherichiacoli's translational machinery so as to increase the suppressionefficiency of the tRNA. It is noteworthy that the G37A mutation wasfound in tRNAs selected from both the anticodon-loop and all-looplibrary. This mutation is consistent with previous studies that showingthat adenine at position 37 enhances amber suppression efficiency. See,e.g., M. Yarus, Translational efficiency of transfer RNA's: Use of anexpanded anticodon, Science 218:646-652 (1982); D. Bradley, J. V. Park,L. Soll, tRNA2Gln Su+2 mutants that increase amber suppression, J.Bacteriol. 145:704-712 (1981); and, L. G. Kleina, J. Masson, J.Normanly, J. Abelson, J. H. Miller, Construction of Escherichia coliamber suppressor tRNA genes. II. Synthesis of additional tRNA genes andimprovement of suppressor efficiency, J. Mol. Biol. 213:705-717 (1990).Fechter et al. recently reported that the complete identity set forMethanococcus jannaschii tRNATyr is six nucleotides (C1G72, A73, andanticodon G34U35A36). See P. Fechter, J. Rudinger-Thirion, M. Tukalo, R.Giegé, Major tyrosine identity determinants in Methanococcus jannaschiiand Saccharomyces cerevisiae tRNATyr are conserved but expresseddifferently, Eur. J. Biochem. 268:761-767 (2001). The presence of C32and T60 in all selected mutant suppressors therefore is not required forrecognition by Methanococcus jannaschii TyrRS. All Escherichia colitRNAs have T at position 60 except four tRNAs which have C. See, M.Sprinzl, C. Horn, M. Brown, A. Loudovitch, S. Steinberg, Compilation oftRNA sequences and sequences of tRNA genes, Nucleic Acids Res.26:148-153 (1998). Based on the crystal structure of yeast tRNAPhe,nucleotide 60 does not interact with other nucleotides. See J. L.Sussman, S. R. Holbrook, R. W. Warrant, G. M. Church, S. H. Kim, Crystalstructure of yeast phenylalanine transfer RNA. J. Crystallographicrefinement, J. Mol. Biol. 123:607-630 (1978). Thus, T60 may maintain theshape of the TC loop for productive interaction with the Escherichiacoli translational machinery. The change of the TC loop structure mayaffect translational fidelity, as the insertion of a nucleotide betweenT60 and the conserved C61 enables a glycine tRNA to shift reading frame.See, D. J. O'Mahony, B. H. Hims, S. Thompson, E. J. Murgola, J. F.Atkins, Glycine tRNA mutants with normal anticodon loop size cause 1frameshifting, Proc. Natl. Acad. Sci. USA 86:7979-7983 (1989). The roleof C32 is not obvious—position 32 in Escherichia coli tRNAs includes T,C, and A, and two Escherichia coli tRNATyrs do have C32. As for position17a, only tRNAThr has an A at this position.

All of the selected suppressor tRNAs are poorer substrates forEscherichia coli synthetases relative to the Methanococcus jannaschiitRNATyrCUA, resulting in less mischarging when introduced intoEscherichia coli. These tRNAs can also be stably maintained inEscherichia coli without adverse effects on the growth of host cells.Moreover, they can still be charged efficiently by Methanococcusjannaschii TyrRS. All these properties make the mutant suppressor tRNAtogether with the Methanococcus jannaschii TyrRS a robust orthogonaltRNA-synthetase pair for the selective incorporation of unnatural aminoacids into proteins in vivo. The J17 mutant suppressor tRNA and anengineered mutant TyrRS has been used to deliver O-methyl-L-tyrosine inresponse to a TAG codon with a fidelity rivaling that of the common 20amino acids. See, L. Wang, A. Brock, B. Herberich, P. G. Schultz,Expanding the genetic code of Escherichia coli, Science, 292:498-500(2001).

Example 2 Mutating TyrRS so that it Charges the mutRNA Tyr/CUA with anUnnatural Amino Acid, O-Methyl-L-Tyrosine

A unique transfer RNA (tRNA)-aminoacyl tRNA synthetase pair has beengenerated that expands the number of genetically encoded amino acids inEscherichia coli. When introduced into Escherichia coli, this pair leadsto the in vivo incorporation of the synthetic amino acidO-methyl-L-tyrosine, added exogenously to the growth medium, intoprotein in response to an amber nonsense codon. The fidelity oftranslation is greater than 99%, as determined by analysis ofdihydrofolate reductase containing the unnatural amino acid. Thisapproach provides a general method for increasing the genetic repertoireof living cells to include a variety of amino acids with novelstructural, chemical and physical properties not found in the commontwenty amino acids.

An orthogonal tRNA/synthetase pair in Escherichia coli can be generatedby importing a pair from a different organism, if cross-speciesaminoacylation is inefficient, and, optionally, the anticodon loop isnot a key determinant of synthetase recognition. One such candidate pairis the tyrosyl tRNA/synthetase pair of Methanococcus jannaschii(Methanococcus jannaschii), an archaebacterium whose tRNATyr identityelements differ from those of Escherichia coli tRNA^(Tyr) (inparticular, the first base pair of the acceptor stem is GC inEscherichia coli and CG in Methanococcus jannaschii), and whose tyrosylsynthetase (TyrRS) has only a minimalist anticodon loop binding domain.See, e.g., B. A. Steer, & P. Schimmel, J. Biol. Chem. 274:35601-6(1999). In addition, the Methanococcus jannaschii TyrRS does not have anediting mechanism, see, e.g., Jakubowski & Goldman, Microbiol. Rev.,56:412 (1992), and therefore should not proofread an unnatural aminoacid ligated to the tRNA. The Methanococcus jannaschii TyrRS efficientlyaminoacylates an amber suppressor tRNA derived from its cognate tRNATyr,see, e.g., Wang, et al., (2000 J. Am. Chem. Soc., supra., but does notaminoacylate Escherichia coli tRNAs, see, e.g., Steer & Schimmel,(1999), supra. Moreover, the Methanococcus jannaschii tRNA_(CUA) ^(Tyr)is a poor substrate for the Escherichia coli synthetases but functionsefficiently in protein translation in Escherichia coli. See, e.g., Wang,et al., (2000 J. Am. Chem. Soc., supra.

To further reduce recognition of the orthogonal tRNA, Methanococcusjannaschii tRNA_(CUA) ^(Tyr), by Escherichia coli synthetases, elevennucleotides of the tRNA that do not interact directly with theMethanococcus jannaschii TyrRS(C16, C17, U17a, U20, C32, G37, A38, U45,U47, A59 and U60) were randomly mutated to generate a suppressor tRNAlibrary. This tRNA library was passed through a negative selection(e.g., suppression of amber mutations in a toxic reporter gene, e.g.,barnase gene), which removes tRNAs that are aminoacylated by Escherichiacoli synthetases, and then a positive selection for tRNAs that areefficiently aminoacylated by Methanococcus jannaschii TyrRS (e.g.,suppression of amber mutations in a reporter gene, e.g., ∃-lactamasegene).

The orthogonal nature of the resulting suppressor tRNAs was tested by anin vivo complementation assay, which is based on suppression of an amberstop codon at a nonessential position (e.g., Ala184) of a reporter geneon a vector, e.g., the TEM-1 β-lactamase gene carried on plasmid pBLAM.Aminoacylation of a transformed suppressor tRNA by any endogenousEscherichia coli synthetase results in cell growth in the presence ofampicillin. Escherichia coli transformed with Methanococcus jannaschiitRNA_(CUA) ^(Tyr) and the reporter construct, pBLAM, survive at 55 μg/mLampicillin. When the best mutant suppressor tRNA (mtRNA_(CUA) ^(Tyr))selected from the library was expressed, cells survived at only 12 μg/mLampicillin; similar values are obtained in the absence of any suppressortRNA. The mutant suppressor tRNA contained the following nucleotidesubstitutions: C17A, U17aG, U20C, G37A, and U47G. When the Methanococcusjannaschii TyrRS is coexpressed with this mtRNAT_(CUA) ^(Tyr), cellssurvive at 440 μg/mL ampicillin. Thus, the mtRNA_(CUA) ^(Tyr), is apoorer substrate for the endogenous synthetases than the Methanococcusjannaschii tRNA_(CUA) ^(Tyr) but is still aminoacylated efficiently bythe Methanococcus jannaschii TyrRS.

To alter the amino acid specificity of the orthogonal TyrRS so that itcharges the mtRNA_(CUA) ^(Tyr) with a desired unnatural amino acid, alibrary of TyrRS mutants was generated and screened. Based on thecrystal structure of the homologous TyrRS from Bacillusstearothermophilus, see, e.g., P. Brick, T. N. Bhat, D. M. Blow, J. Mol.Biol., 208:83 (1988), five residues (Tyr³², Glu¹⁰⁷, Asp¹⁵⁸, Ile¹⁵⁹ andLeu¹⁶²) in the active site of Methanococcus jannaschii TyrRS which arewithin 6.5 Å of the para position of the aryl ring of bound tyrosinewere mutated. See, FIG. 14. These residues were all initially mutated toalanine, and the resulting inactive Ala₅ TyrRS was used as a templatefor polymerase chain reaction (PCR) random mutagenesis with dopedoligonucleotides.

For example, the TyrRS gene was expressed under the control ofEscherichia coli GlnRS promoter and terminator in plasmid pBK-JYRS, apBR322 derived plasmid with kanamycin resistance. Residues Tyr³²,Glu¹⁰⁷, Asp¹⁵⁸, Ile¹⁵⁹ and Leu¹⁶² were substituted with Ala bysite-directed mutagenesis to provide plasmid pBK-JYA5. Eight (8)oligonucleotides with NNK (N=A+T+G+C and K=G+T, and M=C+A), e.g.,oligonucleotides LW157 5′-GGAATTCCATATGGACGAATTTGAAATG-3′ (SEQ IDNO:73), LW164 5′-GTATTTTACCACTTGGTTCAAAACCTATMNNAGCAGATTTTTCATCTTTTTTTCATCTTT TTTTAAAAC-3′ (SEQID NO:74), LW159 5′-TAGGTTTTGAACCAAGTGGTAAAATAC-3′ (SEQ ID NO:75), LW1655′-CATTCAGTGTATAATCCTTATCAAGCTGGAAMNNAC′TTCCATAA ACATATTTTGCCTTTAAC-3′(SEQ ID NO:76), LW161 5′-TCCAGCTTGATAAGGATTATACA CTGAATG-3′ (SEQ IDNO:77), LW167 5′-CATCCCTCCAACTGCAACATCAACGCCMNNATAATGMNNMNNATTAACCTGCATTATTGGATAGATAAC-3′ (SEQ ID NO:78), LW163 5′-GCGTTGATGTTGCAGTTGGAGGGATG-3′ (SEQ ID NO:79), and LW105 5′-AAACTGCAGTTATAATCTCTTTCTAATTGGCTC-3′ (SEQ ID NO:70) (Operon, CA) at the mutation siteswere used for PCR amplification of the Ala₅ TyrRS mutant (pBK-JYA5) andligated back into the NdeI-PstI-digested pBK-JYA5 to afford the TyrRSlibrary. The ligated vectors were transformed into Escherichia coliDH10B competent cells to yield a library of 1.6×10⁹ colony forming unit(cfu). The TyrRS genes from 40 randomly picked colonies were sequencedto confirm that there was no base bias at the randomized NNK positionsand no other unexpected mutations. The library was amplified bymaxiprep, and supercoiled DNA was used to transform the selection strainpYC-J17.

A positive selection was then applied to the library of mutatedorthogonal TyrRS that is based on suppression of an amber stop codon ata nonessential position (e.g., Asp112) in the chloramphenicolacetyltransferase (CAT) gene. See, e.g., M. Pastrnak, T. J. Magliery, P.G. Schultz, Helv. Chim. Acta, 83:2277 (2000). Cells transformed with themutant TyrRS library and mtRNA_(CUA) ^(Tyr), gene were grown in mediacontaining the unnatural amino acid and selected for their survival inthe presence of various concentrations of chloramphenicol. If a mutantTyrRS charges the orthogonal mtRNA_(CUA) ^(Tyr) with any amino acid,either natural or unnatural, the cell produces CAT and survives. Thesurviving cells were then grown in the presence of chloramphenicol andin the absence of the unnatural amino acid. Those cells that did notsurvive, e.g., which encode mutant TyrRS's that charge the orthogonalmtRNA_(CUA) ^(Tyr) with an unnatural amino acid, were isolated from areplica plate supplemented with the unnatural amino acid. The mutantTyrRS genes were isolated from these cells, recombined in vitro by DNAshuffling, and transformed back into Escherichia coli for further roundsof selection with increasing concentrations of chloramphenicol.

A tyrosine analogue with the para hydroxyl group substituted with themethoxy group was used in the selection. Optionally, other tyrosineanalogues can also be used in selection, e.g., tyrosine analogues withdifferent functional groups at the para position of the aryl ring(acetyl, amino, carboxyl, isopropyl, methyl, O-methyl and nitro, etc.).For example, the gene encoding mtRNA_(CUA) ^(Tyr) was expressed inEscherichia coli DH10B cells under the control of the lpp promoter andrrnC terminator in plasmid pYC-J17, a pACYC184 derivative that alsoencodes the Asp₁₁₂ TAG CAT mutant. Supercoiled DNA encoding the TyrRSlibrary was transformed into Escherichia coli DH10B competent cellscontaining pYC-J17 to yield a library of size greater than 3×10⁹ cfu,ensuring complete coverage of the original library. Cells were thenplated on minimal media plates containing 1% glycerol and 0.3 mM leucine(GMML) with 17 μg/mL tetracycline (Tet), 25 μg/mL kanamycin (Kan), 50μg/mL of chloramphenicol (Cm), and 1 mM unnatural amino acid. Afterincubation at 37° C. for 44 hours, colonies on plates supplied withO-methyl-L-tyrosine were pooled, plasmids were isolated and retransforminto Escherichia coli DH10B competent cells containing pYC-J17, and thetransformed cells were positively selected on 50 μg/mL of Cm. Colonies(96) were individually picked from the plate, diluted into 100 μL ofliquid GMML media, and streaked onto two sets of Kan/Tet GMML plateswith various concentration of Cm. No O-methyl-L-tyrosine was added toplate set 1 and the concentration of Cm was varied from 10-25 μg/mL;plate set 2 contained 1 mM O-methyl-L-tyrosine and 50 μg/mL of Cm.Replicates of colonies that did not grow on 15 μg/mL of Cm in plate set1 were picked from plate set 2. Plasmids containing the TyrRS gene werepurified and recombined in vitro by DNA shuffling using Stemmer'sprotocol with the exception of 10 mM Mn2+ instead of Mg2+ in thefragmentation reaction. See, W. P. C. Stemmer, Nature 370, 389-91(1994); and, I. A. Lorimer, I. Pastan, Nucleic Acids Res. 23, 3067-8(1995). The library was then religated into predigested pBK-JYA5 vectorto afford a second generation TyrRS library with a typical size of 8×10⁸to 3×10⁹ cfu. Thirty randomly selected members from the library weresequenced. The mutagenic rate introduced by DNA shuffling was 0.35%.This library was transformed into the selection strain for the nextround of selection followed by shuffling. The concentration of Cm in thepositive selection and in plate set 2 was raised to 80 μg/mL for thesecond round and 120 μg/mL for the third round; the concentration of Cmin plate set 1 was unchanged. After three rounds of DNA shuffling,colonies began to grow on 20-25 μg/mL Cm in plate set 1, indicating thatthe TyrRS mutants were accepting natural amino acids as substrates.Therefore, the best clone selected after two rounds of DNA shuffling wascharacterized in detail.

Two rounds of selection and DNA shuffling were carried out and a clonewas evolved whose survival in chloramphenicol was dependent on theaddition of 1 mM O-methyl-L-tyrosine to the growth media. In the absenceof O-methyl-L-tyrosine, cells harboring the mutant TyrRS were not viableon minimal media plates containing 1% glycerol, 0.3 mM leucine (GMML),and 15 μg/mL of chloramphenicol. Cells were able to grow on GMML plateswith 125 μg/mL chloramphenicol in the presence of 1 mMO-methyl-L-tyrosine. Similar results were obtained in liquid GMML. As acontrol, cells with the mtRNA_(CUA) ^(tyr) and the inactive Ala₅ TyrRSdid not survive at the lowest concentration of chloramphenicol used,either in the presence or absence of 1 mM O-methyl-L-tyrosine. See FIG.14. Addition of 1 mM O-methyl-L-tyrosine itself does not significantlyaffect the growth rate of Escherichia coli.

Analysis of the sequence of the mutant TyrRS that charges themtRNA_(CUA) ^(Tyr) with O-methyl-L-tyrosine revealed the followingmutations: Tyr³²→Gln³², Asp¹⁵⁸→Ala¹²⁸, Glu¹⁰⁷→Thr¹⁰⁷, and Leu¹⁶²→Pro¹⁶².See FIG. 14. Based on the x-ray crystal structure of the homologous B.stearothermophilus TyrRS, the loss of the hydrogen-bonding networkbetween Tyr³², Asp¹⁵⁸ and substrate tyrosine can disfavor binding oftyrosine to the mutant TyrRS. Indeed, mutation of Asp¹⁷⁶ (whichcorresponds to Asp¹⁵⁸ in Methanococcus jannaschii) of B.stearothermophilus TyrRS yields inactive enzyme. See, e.g., G. D. P.Gray, H. W. Duckworth, A. R. Fernst, FEBS Lett. 318:167 (1993). At thesame time, the Asp¹⁵⁸→Ala¹⁵⁸ and Leu¹⁶²→Pro¹⁶² mutations create ahydrophobic pocket that allows the methyl group of O-methyl-L-tyrosineto extend further into the substrate-binding cavity. Other importantcatalytic residues in the active site, which bind to the ribose or thephosphate group of the adenylate, were unchanged after two rounds of DNAshuffling.

Kinetics of adenylate formation of O-methyl-L-tyrosine and tyrosine withadenosine triphosphate (ATP) catalyzed by the mutant TyrRS were analyzedin vitro using a pyrophosphate-exchange assay at 37° C. For example, themutant TyrRS gene with six histidines at its C-terminus was cloned intoplasmid pQE-60 (Qiagen, Calif.) to generate plasmid pQE-mJYRS. Proteinwas purified by immobilized metal affinity chromatography according tomanufacture's protocol (Qiagen, Calif.). Pyrophosphate (PPi) exchangewas carried out at 37° C. in a reaction mixture containing 100 mMTrisHCl (pH7.5), 10 mM KF, 5 mM MgCl₂, 2 mM ATP, 2 mM NaPPi, 0.1 mg/mLbovine serum albumin, approximately 0.01 μCi/μL [³²P]NaPPi, and variousconcentrations of tyrosine or O-methyl-L-tyrosine. Reactions wereinitiated with the addition of the purified mutant TyrRS, and aliquotswere periodically taken and quenched with 0.2 M NaPPi, 7% perchloricacid, and 2% activated charcoal. The charcoal was filtered and washedwith 10 mM NaPPi (pH2), then measured by scintillation counting todetermine the ³²P levels in charcoal-adsorbed ATP. Values of k_(cat) andK_(m) were calculated by direct fitting of the Michaelis-Menten equationusing nonlinear regression analysis.

The Michaelis constant (K_(m)) for tyrosine (5833+/−9020A) isapproximately 13-fold higher than that for O-methyl-L-tyrosine (443+/−93μM), and the catalytic rate constant (k_(cat)) for tyrosine(1.8+/−0.2×10⁻³ s⁻¹) is eightfold less than that for O-methyl-L-tyrosine(14+/−1×10⁻³ s⁻¹). Thus, the value of k_(cat)/K_(m) of the mutant TyrRSfor O-methyl-L-tyrosine is about 100-fold higher than that of tyrosine.The physiological concentration of tyrosine in Escherichia coli is about80 μM, which is far below K_(m) value (5833 μM) of the mutant TyrRS fortyrosine. Presumably, the concentration of O-methyl-L-tyrosine in cellsis comparable or greater than the K_(m) (443 μM).

This example shows that it is possible to augment the proteinbiosynthetic machinery of Escherichia coli to accommodate additionalgenetically encoded amino acids. The ability to introduce novel aminoacids into proteins directly in living cells will provide new tools forstudies of protein and cellular function and can lead to generation ofproteins with enhanced properties compared to a naturally occurringprotein. The methods described here can be applied to other amino acidswith novel spectroscopic, chemical, structural or the like properties.The Escherichia coli ribosome has been shown to be able to incorporateamino acids with a wide array of side chains into proteins using invitro protein synthesis. See, e.g., C. J. Noren, S. J. Anthony-Cahill,M. C. Griffith, P. G. Schultz, Science 244, 182-8 (1989). Additionalorthogonal tRNA/synthetase pairs, see, e.g., D. R. Liu, P. G. Schultz,Proc. Natl. Acad. Sci. USA 96, 4780-5 (1999); and, A. K. Kowal, C.Kohrer, U. L., RajBhandary, Proc. Natl. Acad. Sci, U.S.A., 98:2268(2001), as well as four base codons, see, e.g., T. J. Magliery, J. C.Anderson, P. G. Schultz, J. Mol. Biol. 307:755 (2001); and, B. Moore, B.C. Persson, C. C. Nelson, R. F. Gesteland, J. F. Atkins, J. Mol. Biol.,298:195 (2000), and other selector codons described herein, can furtherexpand the number and scope of amino acids that can be incorporated.Orthogonal pairs for eukaryotic cells can also be generated by themethods provided herein.

See also corresponding patent application “In vivo Incorporation ofUnnatural Amino Acids” attorney docket number 54-000120PC/US (U.S. Ser.No. 10/126,927, now U.S. Pat. No. 7,045,337) which is incorporatedherein by reference. This application describes an example of thegeneration of an O-methyl-L-tyrosine mutant of dihydrofolate reductase(DHFR) using the above-described system.

Example 3 Mutating TyrRS so that it Charges the mutRNA Tyr/CUA with anUnnatural Amino Acid, L-3-(2-Napthyl)Alanine

This example provides another orthogonal pair that can be used toincorporate a second unnatural amino acid, L-3-(2-Napthyl)alanine intoproteins in an organism, e.g., Escherichia coli. An example of themethods used to generate the orthogonal pair that incorporates theunnatural amino acid into proteins is described below. More detailsdescribing the incorporation of the unnatural amino acid into a proteincan be found in corresponding patent application “In vivo incorporationof unnatural amino acid” attorney docket number 54-000120PC/US (U.S.Ser. No. 10/126,927, now U.S. Pat. No. 7,045,337) incorporated herein byreference.

An amber stop codon and its corresponding orthogonal amber suppressortRNA, mu tRNA_(CUA) ^(Tyr), were selected to encode an unnatural aminoacid. As described above, and see Wang & Schultz, Chem. Biol. 8:883-890(2001). The Methanococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) wasused as the starting point for the generation of an orthogonalsynthetase with unnatural amino acid specificity. This TyrRS does notaminoacylate any endogenous Escherichia coli tRNAs, see, e.g., Steer &Schimmel, J. Biol. Chem., 274:35601-35606 (1999), but aminoacylates themu tRNA_(CUA) ^(Tyr) with tyrosine. See, e.g., Wang, Magliery, Liu,Schultz, J. Am. Chem. Soc., 122:5010-5011 (2000).L-3-(2-naphthyl)-alanine was chosen for this study since it represents asignificant structural perturbation from tyrosine and may have novelpacking properties. To change the amino acid specificity of the TyrRS sothat it charges the mu tRNA_(CUA) ^(Tyr) with L-3-(2-naphthyl)-alanineand not any common 20 amino acids, a library of Methanococcus jannaschiiTyrRS mutants was generated and screened. On the basis of an analysis ofthe crystal structure of the homologous TyrRS from Bacillusstearothermophilus, see, Brick, Bhat, Blow, J. Mol. Biol., 208:83-98(1989), five residues (Tyr³², Asp¹⁵⁸, Ile¹⁵⁹, Leu¹⁶², and Ala¹⁶⁷) in theactive site of Methanococcus jannaschii TyrRS that are within 7 Å of thepara position of the aryl ring of tyrosine were mutated. See FIG. 15. Nosynthetases specific for L-3-(2-naphthyl)alanine were selected from themutant TyrRS library reported in Wang, Brock, Herberich, Schultz,Science, 292:498-500 (2001). To reduce the wild-type synthetasecontamination in the following selection, these residues (except Ala¹⁶⁷)were first all mutated to alanine. The resulting inactive Ala₅ TyrRSgene was used as a template for polymerase chain reaction (PCR) randommutagenesis with oligonucleotides bearing random mutations at thecorresponding sites.

The mutant TyrRS library was first passed through a positive selectionbased on suppression of an amber stop codon at a nonessential position(Asp¹¹²) in the chloramphenicol acetyltransferase (CAT) gene. Cellstransformed with the mutant TyrRS library and the mu tRNA_(CUA) ^(Tyr)gene were grown in minimal media containing 1 mML-3-(2-naphthyl)-alanine and 80 mg/mL chloramphenicol. Cells can surviveonly if a mutant TyrRS aminoacylates the mu tRNA_(CUA) ^(Tyr) witheither natural amino acids or L-3-(2-naphthyl)-alanine. The survivingcells were then grown in the presence of chloramphenicol and the absenceof the unnatural amino acid. Those cells that did not survive mustencode a mutant TyrRS that charges the mu tRNA_(CUA) ^(Tyr) withL-3-(2-naphthyl)-alanine, and were picked from a replica plate suppliedwith the unnatural amino acid. After three rounds of positive selectionfollowed by a negative screen, four TyrRS's were characterized using anin vivo assay based on the suppression of the Asp¹¹²TAG codon in the CATgene.

TABLE 4 In vivo chloramphenicol acetyltransferase assay of mutantTyrRS.^(a) IC₅₀ (μg/mL of chloramphenicol) Mutant TyrRS NoL-3-(2-naphthyl)-Ala Add L-3-(2-naphthyl)-Ala no TyrRS 4 4 wt TyrRS 240240 After selection S1-TyrRS 30 120 S2-TyrRS 30 120 S3-TyrRS 25 110S4-TyrRS 35 100 After DNA shuffling SS12-TyrRS 9 150 ^(a)A pYC-J17plasmid was used to express the mu _(tRNA) _(CUA) _(Tyr) gene and thechloramphenicol acetyltransferase gene with an amber stop codon atAsp112. A pBK plasmid was used to express TyrRS, and was cotransformedwith pYC-J17 into Escherichia coli DH10B. Cell survival on GMML plateswas titrated in the presence of different concentrations ofchloramphenicol.

In the absence of L-3-(2-naphthyl)-alanine, cells expressing theselected TyrRS and the mu tRNA_(CUA) ^(Tyr) survived in 25 to 35 μg/mLchloramphenicol on minimal media plates containing 1% glycerol and 0.3mM leucine (GMML plate); in the presence of L-3-(2-naphthyl)-alanine,cells survived in 100 to 120 μg/mL chloramphenicol on GMML plates.Compared to the IC₅₀ value in the absence of any TyrRS (4 μg/mLchloramphenicol), these results indicate that the selected TyrRS'saccept L-3-(2-naphthyl)-alanine, but also still charge natural aminoacids to some degree. See Table 4 above.

To further reduce the activity of the mutant TyrRS toward natural aminoacids, one round of DNA shuffling was carried out using the above fourmutant genes as templates. The resulting mutant TyrRS library was passedthrough two additional rounds of positive selections and negativescreens. One mutant TyrRS(SS12-TyrRS) was evolved, whose activity fornatural amino acids was greatly reduced (IC₅₀=9 μg/mL chloramphenicol)while its activity toward L-3-(2-naphthyl)-alanine was enhanced(IC₅₀=150 μg/mL chloramphenicol). See Table 4.

The evolved SS12-TyrRS has the following mutations: Tyr³²→Leu³²,Asp¹⁵⁸→Pro¹⁵⁸, Ile¹⁵⁹→Ala¹⁵⁹, Leu¹⁶²→Gln¹⁶², and Ala¹⁶⁷→Val¹⁶⁷. See FIG.15. Based on the crystal structure of the homologous B.stearothermophilus TyrRS, the mutations of Tyr³²→Leu³² and Asp¹⁵⁸→Pro¹⁵⁸can result in the loss of hydrogen bonds between Tyr³², Asp¹⁵⁸, and thenative substrate tyrosine, thus disfavoring the binding of tyrosine toSS12-TyrRS. Most residues are mutated to amino acids with hydrophobicside chains, which are expected to favor binding ofL-3-(2-naphthyl)-alanine. The crystal structure of the wild-typeMethanococcus jannaschii TyrRS and the evolved SS12-TyrRS can bedetermined by available methods.

The mu tRNA_(CUA) ^(Tyr)/SS12-TyrRS pair was capable of selectivelyinserting L-3-(2-naphthyl)-alanine into proteins in response to theamber codon with fidelity rivaling that of the natural amino acids basedon cell growth, protein expression and mass spectrometry examplesdescribed herein and in corresponding application “In vivo incorporationof unnatural amino acids” attorney docket number 54-000120PC/US (U.S.Ser. No. 10/126,927, now U.S. Pat. No. 7,045,337). See also, Wang,Brock, and Schultz, Adding L-3-(2-Naphthyl)alanine to the genetic codeof E. coli, J. Am. Chem. Soc., (2002) 124(9):1836-7. This result, whichinvolves an amino acid that is structurally distinct from tyrosine,confirms that the methods described herein are generalizable to avariety of unnatural amino acids.

Example 4 Mutating TyrRS so that it Charges the mutRNA Tyr/CUA andScreening for the Mutated TyrRS with the Desired Properties by OtherMethods, e.g., FACs and Phage Display and Panning

Orthogonal pairs can also be selected by using reporter genes andproteins as described above, along with in vivo FACS screening, antibodydetection, in vitro phage display and panning, or the like. See, Wang &Schultz, Expanding the genetic code, Chem. Commun., 1:1-11 (2002).

For example, a general fluorescence-activated cell sorting (FACS) basedscreen has been developed with, e.g., green fluorescent protein (GFP) asthe reporter, to screen for synthetases. See FIG. 16, Panel A, and PanelB Synthetase activity is reported by suppression of the selector codon,e.g., an amber stop codon (TAG) within T7 RNA polymerase, which drivesthe expression of GFP. See, e.g., FIG. 26 for another example ofselection/screening methods of the invention. Only when the amber codonsare suppressed can cells produce functional T7 RNA polymerase andexpress GFP, rendering cells fluorescent. In the positive screen,fluorescent cells are collected which encode active synthetases chargingthe orthogonal tRNA with either natural or unnatural amino acids. Theselected cells are then diluted and grown in the absence of theunnatural amino acid, and then sorted by FACS for cells withoutfluorescence, e.g., that express synthetases with specificities forunnatural amino acids only. FIG. 17, Panel A, Panel B Panel C and PanelD illustrates suppression of a selector codon, e.g., an amber codon,using glutamine synthetase. By setting the collection threshold of thefluorescence intensity, the stringency of both positive and negativescreen can be conveniently controlled.

A direct positive selection specific for a particular unnatural aminoacid has also been developed which exploits the high affinity of amonoclonal antibody for an unnatural amino acid displayed on a phagesurface. See FIG. 18. See, M. Pastrnak and P. G. Schultz, Bioorg. Med.Chem., 9:2373 (2001). For example, a C3 peptide with an amber mutationis fused to the N-terminus of VSCM13 phage coat protein pIII, such thatphage production requires suppression of the amber stop codon. Cellsharboring a phagemid that expresses an orthogonal suppressor tRNA and asynthetase library are infected with the C3TAG phage. An activesynthetase results in suppression of C3TAG and display of its cognateamino acid on the phage surface. The phage pool is then incubated withimmobilized monoclonal antibodies directed against the unnatural aminoacid to isolate only those phage carrying the synthetase specific forthe unnatural amino acid. In a simulated selection, phage displaying Aspwere enriched over 300-fold from a pool of phage displaying Asn usingantibodies raised against the Asp-containing epitope.

Several in vitro screen methods can also be used. In one such method, alibrary of mutant synthetases is displayed on the phage, and the phageparticles are panned against immobilized aminoalkyl adenylate analogs ofthe aminoacyl adenylate intermediate. See FIG. 19. For example,Methanococcus jannaschii TyrRS was fused to the pill coat protein of M13phage. This phage was enriched 1000-fold over a control phage displayingan unrelated antibody after panning against the aminoalkyl adenylateanalog of tyrosyl adenylate. Given that only 0.1 to 1% of the startingTyrRS phage population displays the TyrRS protein, the actual enrichmentfactor can be as high as 10⁵ to 10⁶.

Example 5 Generating an Archaeal Leucyl-tRNA Synthetase Pair

A leucyl-tRNA synthetase from an archaebacterium, Methanobacteriumthermoautotrophicum, was identified that can aminoacylate amber andframeshift suppressor tRNAs derived from archaeal leucyl tRNAs, but doesnot aminoacylate any tRNAs native to Escherichia coli. Using a selectionstrategy described in the present invention, highly active tRNAsubstrates were identified that are selectively charged by thesynthetase. Mutant libraries of synthetases can be generated andselected for that are capable of selectively charging unnatural aminoacids.

β-lactamase reporter genes were constructed with amber codons andsuppressor tRNAs derived from five different archael leucyl tRNAs forwhich the anticodon was replaced with a CUA anticodon to make ambersuppressor tRNAs. Seven different leucyl tRNA synthetases were clonedand were cotransformed with reporter constructs. Three synthetases gavehigher levels of survival on ampicillin in the presence of thesynthetase than controls lacking synthetase, and these systems wereexamined further. See, FIG. 20.

The next step involved determination of a synthetase that charges thesuppressor tRNA without interacting with host tRNA. The two chosensystems, Methanobacterium thermoautotrophicum and Methanococcusjannaschii were expressed, and aminoacylation was performed in vitro onpurified tRNA from Halobacterium as a positive control, and forEscherichia coli total tRNA. It was found that the Methanococcusjannaschii synthetase was able to effectively charge Escherichia colitRNA, but the Methanobacterium thermoautotrophicum synthetase wasspecific towards the Halobacterium tRNA.

Further improvements were made to increase the efficiency of thesuppression system. The A³⁷ site of the anticodon loop was a G³⁷ in theleucyl tRNA synthetases. This mutation has been shown to be a negativedeterminant against aminoacylation by non-cognate synthetases in variouseukaryotic cells and Halobacterium, and also a positive determinate foraminoacylation in yeast, but not in Halobacterium. A³⁷ was also shown tobe a key requirement for efficient suppression. The anticodon loop wasrandomly mutagenized and selected for more efficient suppression.Mutating G³⁷ to A, resulted in a more efficient suppressor, which couldsuppress 20 fold higher concentrations of ampicillin compared to theun-mutated version. See, FIG. 21.

To improve the tRNA so that is not preferentially charged by othersynthetases in Escherichia coli, the acceptor stem of the tRNA wasrandomly mutagenized. A positive/negative selection was used to identifytRNAs that would not be charged in the absence of Methanobacteriumthermoautotrophicum RS.

Amongst the selected mutated tRNAs observed, all conserved thediscriminator base, A⁷³, which has been shown in all previous systems tobe a critical positive determinate for leucyl aminoacylation. Alsoconserved was a C³:G⁷⁰ base pair amongst all hits that had improvedorthogonality. The best mutant tRNA observed gave about a 3-folddecrease in aminoacylation without synthetase and actually an increasein suppression in the presence of Methanobacterium thermoautotrophicumRS.

Variants were also made that could suppress four-base codons instead of,e.g., three base codons. Four base codons offer the possibility ofdecoding the genetic code four bases at a time, for which 256 thingscould be encoded rather than 3 at a time, where only 64 amino acids canbe encoded. The difficulty with using four-base codons is that theyrequire expansion of the anticodon loop for the tRNA, a perturbationwhich most systems are unlikely to accept. However, a first generationAGGA suppressor for the leucyl system was identified. This was generatedby randomly mutagenizing the anticodon loop with 8 bases and performingselection with an AGGA-β-lactamase reporter system. See FIG. 22.

The editing mechanism of the synthetase was also mutated to eliminatethe editing function. The leucyl system, like several other synthetaseshas (at least) two active sites. One site performs activation of theamino acid with ATP to form an enzyme bound aminoacyl adenylate incomplex with the synthetase, and then transfer of the amino acid ontothe 3′ terminus of the tRNA. A second site, however, is able tohydrolyze the amino acid from the tRNA if it is not leucine. The leucinesystem is known to perform this post-transfer editing function formethionine and isoleucine, and it optionally does this to unnaturalamino acids as well.

Initially, the editing domain was deleted. The editing domain wasreplaced with a library of 6 tandem random amino acids. A positiveselection was used, which was based on suppression of a stop codon inβ-lactamase. Many functional synthetases were obtained, but upon tryingto purify the synthetases, no material in any cases could be detected,and all of these synthetases displayed a temperature sensitive phenotypesuggesting that the deletion of the editing domain resulted in a lessstable protein.

Next, point mutations were made in the editing domain. The catalyticcore of the editing domain is well conserved across species and even fordifferent amino acids, at least for the family of branched chain aminoacids. Several of these conserved sites have previously been mutated,for example a T→P mutation, and found to knock out editing function.Mutants of Methanobacterium thermoautotrophicum RS were constructed thatwere similar to several known mutants, and also a 20 member NNK libraryderived from T214 was made. Proteins were expressed and examined invitro for aminoacylation with leucine and methionine. None of thepreviously identified mutations were transferable to our system, but adesirable mutation was identified from the T214 library. Two mutantswere identified that were capable of charging with leucine, T214S andT214Q. Of these mutations, only T214Q was capable of chargingmethionine. The T214S mutant apparently retains the ability to edit outmethionine whereas the Gln mutant has lost this function.

A library was then designed based on the crystal structure that has beensolved for the Thermus thermophilus leucyl synthetase. The leucine sidechain of the leucine aminoalkyl adenylate analog adenosine inhibitor wasbound in the active site. Six sites surrounding the leucine sidechain-binding pocket were replaced with randomized amino acids to createa larger library. The synthetases from this library can then bescreened, e.g., by performing positive/negative double sieve selections,to identify synthetases capable of charging unnatural amino acidsselectively.

Example 6 Identification of tRNAs that Efficiently Suppress Four-BaseCodons

A combinatorial approach was used to identify mutated tRNAs thatefficiently suppress four-base codons. See, T. J. Magliery, J. C.Anderson and P. G. Schultz, J. Mol. Biol., 307:755 (2001). A reporterlibrary was constructed in which a serine codon in the ∃-lactamase genewas replaced by four random nucleotides. A mutated tRNA, e.g.,suppressor tRNA, suppressor library was then generated that consists ofderivatives of Escherichia coli with the anticodon loop (7 nt) replacedwith eight or nine random nucleotides. When these two libraries arecrossed, an appropriate frameshift suppressor tRNA that decodes thefour-base sequence as a single codon results in translation offull-length ∃-lactamase, rendering the cells resistant to ampicillin.Survival at higher concentrations of ampicillin indicates that thecorresponding tRNA has higher suppression efficiency for the four-basecodon. Using this selection, four quadruplet codons AGGA, CUAG, UAGA,and CCCU and their cognate suppressor tRNAs were identified that decodeonly the canonical four-base codon with efficiencies close to that ofnatural triplet codon suppressors. Novel five- and six-base codonsuppressors have also been selected using this strategy. See, Anderson,Magliery, Schultz, Exploring the Limits of Codon and Anticodon Size,Chemistry & Biology, 9:237-244 (2002). These extended codons, some ofwhich are newly identified, can be useful for the incorporation ofmultiple unnatural amino acids in vitro and for in vivo proteinmutagenesis.

Example 7 Generation of an Orthogonal tRNA-Synthetase forp-Aminophenylalanine

To generate an orthogonal synthetase pair for p-aminophenylalanine(pAF), the Methanococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) andmutant tyrosine amber suppressor tRNA (TyrCUA mutRNA) pair were used asa starting point. See, Wang, L., Magliery, T. J., Liu, D. R. & Schultz,P. G. A new functional suppressor tRNA/aminoacyl-tRNA synthetase pairfor the in vivo incorporation of unnatural amino acids into proteins. J.Am. Chem. Soc. 122:5010-5011 (2000); and, Wang, L. & Schultz, P. G.Chem. and Biol. 8:883 (2001). The pAF specific synthetase (pAFRS) wasgenerated by modifying the amino acid specificity of the Methanococcusjannaschii TyrRS to accept pAF and not any of the common twenty aminoacids. A combination of positive selections and negative screens wasused to identify the pAFRS enzyme from a library of TyrRS variants 12containing random amino acids at five positions (Tyr³², Glu¹⁰⁷, Asp¹⁵⁸,Ile¹⁵⁹, and Leu¹⁶²). See, Wang, L., Brock, A., Herberich, B. & Schultz,P. G. Expanding the genetic code of Escherichia coli. Science292:498-500 (2001). A single reporter plasmid was used for bothselection and screening. For example, the reporter plasmid ispREP(2)/YC-JYCUA, which contains the genes for CAT, T7 RNA polymerase,GFP, and _(TyrCUA)mutRNA, and a selectable marker for Tet resistance.The CAT gene contains a TAG codon substitution at position D112. The T7RNA polymerase gene contains a seven-amino acid N-terminal leaderpeptide and TAG substitutions at M1 and Q107.

The positive selection is based on suppression of a TAG codon at apermissive position within the chloramphenicol acetyltransferase (CAT)gene by either pAF or an endogenous amino acid. See, e.g., Wang et al.(2001), supra; and, Pastrnak, M., Magliery, T. J. & Schultz, P. G. A neworthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair for evolvingan organism with an expanded genetic code. Helvetica Chemica Acta83:2277 (2000). Cells containing the TyrRS library and reporter plasmidwere grown in liquid culture containing pAF and selected for survival inthe presence of chloramphenicol (Cm). For example, for the positiveselection, cells were grown in GMML minimal media containing 35 μg/mlKn, 25 μg/ml Tet, 75 μg/ml Cm, and 1 mM pAF (Sigma).

The negative screen is based on the inability to suppress in the absenceof pAF two TAG stop codons at permissive positions within the T7 RNApolymerase gene. Expression of full length T7 RNA polymerase drivesexpression of green fluorescent protein (GFP). Cells from the positiveselection were grown in the absence of pAF and Cm, and then screenedusing fluorescence activated cell sorting (FACS) for a lack offluorescence. For example, for the negative screen, cells were grown inGMML media containing 35 μg/ml Kn, 25 μg/ml Tet, and 0.002% arabinose.FACS was carried out using a BDIS FACVantage TSO cell sorter with aCoherent Enterprise II ion laser. The excitation wavelength was 351 nmand emission was detected using a 575/25 nm bandpass filter. Collectedcells were diluted into at least 10 volumes of LB, containing Tet andKn, and grown to saturation.

The desired pAFRS was identified following two rounds of positiveselection in liquid media, one round of negative screening, anotherround of positive selection in liquid media, and one round of positiveselection on plates. The pAFRS enzyme contains five mutations relativeto the wild type TyrRS (Y32T, E107T, D158P, I159L, and L162A). In theabsence of pAF, the IC₅₀ of cells expressing the selected pAFRS andreporter plasmid was 10 μg/ml Cm on GMML minimal media plates. The IC₅₀was 120 μg/ml Cm with 1 mM pAF. Thus, pAF is selectively suppressing theUAG codon.

Example 8 Evolution of an Aminoacyl-tRNA Synthetase UsingFluorescence-Activated Cell Sorting.

A FACs based screening system was used to rapidly evolve three highlyselective synthetase variants that accept amino-, isopropyl-, orallyl-containing tyrosine analogues. The system included a multipurposereporter plasmid used for application of both positive and negativeselection pressure and for the facile and quantitative evaluation ofsynthetase activity. A chloramphenicol acetyl transferase (CAT) markerallowed positive selection for activity of the M. jannaschiityrosyl-tRNA synthetase (TyrRS). A T7 polymerase/GFP reporter systemallowed assessment of synthetase activity within cells grown in both thepresence and absence of an unnatural amino acid. Fluorescence activatedcell sorting (FACS) was used to screen against synthetase variants thataccept natural amino acids, while visual and fluorimetric analyses wereto assess synthetase activity qualitatively and quantitatively,respectively.

Design of an amplifiable fluorescence reporter system. Efforts todevelop a versatile screening system for the assessment of synthetaseactivity in living cells initially arose out of a desire for a greaterdegree of control over the selective pressure applied to populations ofsynthetase variants, especially negative selective pressure. As thesystem was to be used to assess the activities of large numbers ofsynthetase variants, a reporter was sought that would be amenable tohigh-throughput screening. In addition, a reporter that would allow forfacile qualitative and quantitative evaluation of synthetase activitywas desired. To meet these requirements, a fluorescence-based screen wasdesigned. The system was based on the synthetase-dependent production ofGFPuv, a variant of the green fluorescent protein that has beenoptimized for expression in E. coli (see, Crameri, A., Whitehorn, E. A.,Tate, E. & Stemmer, W. P., Nature Biotechnol. 1996, 14, 315-319). Thisfluorophore is amenable to use in FACS and fluorimetry, as well asvisual inspection on plates and in liquid culture. The system wasdesigned such that synthetase-dependent suppression of selector, e.g.,amber nonsense codons would result in the production of a fluorescencesignal. In order to maximize the sensitivity of the reporter, it wasmade amplifiable by placement of the amber codons within the gene for T7RNA polymerase, which was designed to drive expression of the GFPuvreporter gene in analogy to other amplifiable intracellular reportersystems (see, Lorincz, M., Roederer, M., Diwu, Z., Herzenberg, L. A.,Nolan, G. P. Cytometry, 1996, 24, 321-329; and Zlokarnik, G., Negulescu,P. A., Knapp, T. E., Mere, L., Burres, N., Feng, L., Whitney, M.,Roemer, K. & Tsien, R. Y., Science, 1998, 279, 84-88). The T7 RNApolymerase gene was placed under control of the arabinose promoter inorder to allow facile optimization of the production of the RNAtranscript for amber codon-containing T7 RNA polymerase.

Optimization of the T7 RNA polymerase/GFPuv reporter system. Amedium-copy reporter plasmid, pREP, was designed to expressamber-containing T7 RNA polymerase variants under control of thearabinose promoter and the GFPuv gene under control of the T7 promoter(FIG. 17 a). A series of twelve T7 RNA polymerase variants, designed tooptimize synthetase-dependent fluorescence enhancement (FIG. 17 b), wereinserted into pREP to create plasmids pREP(1-12). All variants containedan N-terminal leader sequence of seven amino acids (MTMITVH) and 1-3amber stop codons (TAG). Variants 1-3 contained one, two, and threeamber stop codons, respectively, substituted for the original methionineat position one (M1), just downstream of the leader sequence. Variants4-9 contained an amber codon substituted for D10, R96, Q107, A159, Q169,or Q232, respectively, which were predicted to be located in loopregions of the structure (see, Jeruzalmi, D. & Steitz, T. A., EMBO J.,1998, 17, 4101-4113). Variants 10-12 contained amber stop codonssubstituted at positions M1 and either Q107, A159, or Q232,respectively. Plasmid constructs were evaluated by fluorimetry and flowcytometry of live cells for fluorescence enhancement using a compatibleplasmid containing the orthogonal glutaminyl-tRNA synthetase andGlutamine tRNA_(CUA) from S. cerevisiae. Plasmids pREP(1-12) were foundto provide varying levels of synthetase-dependent fluorescenceenhancement, with the best construct, pREP(10) exhibiting 220-foldgreater fluorescence by fluorimetry (FIGS. 17 c) and ˜400-fold greatermedian fluorescence by cytometry (FIG. 17 d) in cells containing thewild type synthetase versus an inactive mutant. Substitution of avariety of functional groups at positions corresponding to the ambercodons within pREP(10) demonstrate that position 107 within T7 RNApolymerase is highly permissive.

Construction of a multipurpose reporter plasmid. In order to construct amultipurpose plasmid to be used both for selecting and screeningvariants of a M. jannaschii TyrRS, plasmid pREP(10) was combined withplasmid pYC-J17 (see, Wang, L, Brock, A., Herberich, B. & Schultz, P.G., Science, 2001, 292, 498-500) to obtain pREP/YC-JYCUA (FIG. 25 a).Plasmid pREP/YC-JYCUA was assayed for function with a compatible plasmidexpressing a variant of M. jannaschii TyrRS (pBK-mJYRS; Wang, L, Brock,A., Herberich, B. & Schultz, P. G., Science, 2001, 292, 498-500)selective for incorporating O-Methyl-Tyrosine (OMY). Cells containingpREP/YC-JYCUA and pBK-mJYRS, grown in the presence of OMY, exhibited achloramphenicol (Cm) IC₅₀ value of 120 μg/μl, identical to that obtainedusing plasmid pYC-J17, and a fluorescence enhancement of 330-fold forcells grown in the presence versus the absence of OMY, as measured byfluorimetry.

Evolution of the substrate specificity of the M. jannaschii tyrosyl-tRNAsynthetase. Results have shown that the amino acid side chain bindingpocket of the M. jannaschii TyrRS can be evolved to selectivelyaccommodate chemical groups other than the phenol side chain of tyrosine(see, Wang, L, Brock, A., Herberich, B. & Schultz, P. G., Science, 2001,292, 498-500; Wang, L., Brock, A. & Schultz, P. G. J. Am. Chem. Soc.2002, 124, 1836-1837). We sought to further explore the generality ofunnatural amino acid accommodation by M. jannaschii TyrRS by challengingthe enzyme to accept four new functionalities: p-Isopropyl-Phenylalanine(pIF), p-Amino-Phenylalanine (pAF), p-Carboxyl-Phenylalanine (pCF), orO-Allyl-Tyrosine (OAT) (FIG. 25 b). A library of M. jannaschii TyrRSvariants containing randomizations at positions Y32, E107, D158, 1159,and L162 (Wang, L, Brock, A., Herberich, B. & Schultz, P. G., Science,2001, 292, 498-500), residues thought to form the binding pocket for thepara position of the tyrosyl ring, was introduced into cells containingplasmid pREP/YC-JYCUA. These cells, encompassing a library diversity of˜10⁹, were used to begin four evolution experiments to identifysynthetase variants selective for pIF, pAF, pCF, or OAT (FIG. 25 b). Twocycles of positive selection were carried out by allowing the cellcultures to grow to saturation in the presence of Cm and one of the fourunnatural amino acids. Cell aliquots were removed following the secondcycle of positive selection and used to inoculate a new culturecontaining no added amino acid or Cm, and the culture was again allowedto grow to saturation. At this point, cells that fluoresce are likely tocontain synthetase variants that can accept one of the 20 natural aminoacids. Approximately 10⁸ cells from each line were subjected to negativescreening using FACS in order to eliminate natural amino acid-acceptingsynthetase variants. The non-fluorescent cells were collected andamplified through growth to saturation. These amplified cells were usedto inoculate a new culture for a final cycle of positive selection inliquid culture containing unnatural amino acid and Cm. Following growthto saturation, each population of cells was plated on media containing0, 30, 60, or 100 μg/mL Cm and either 0 or 1 mM of the appropriateunnatural amino acid.

Identification and characterization of evolved synthetase variants. Cmplates supplemented with pIF, pAF, and OAT produced 10-100-fold greaternumbers of fluorescent colonies than plates containing no added aminoacid. In contrast, plates for the pCF population produced the samenumber of fluorescent colonies with or without addition of pCF. The tenlargest fluorescent colonies were picked for each of the pIF, pAF, andOAT populations from unnatural amino acid-containing plates and grown tosaturation in liquid media with or without added unnatural amino acid. Aqualitative assessment of fluorescence production was made visually withthe use of a hand-held long-wavelength ultraviolet lamp (FIG. 23 a).

Synthetase variants corresponding to clones producing significantdifferences in fluorescence were sequenced. All ten clones from the pIFand pAF populations had identical sequences, while three differentclones were identified from the OAT population. Amino acid changesoccurred within the five randomized sites in all clones, with theexception of two additional substitutions within the pIF-tRNA synthetase(pIF-RS) variant. The activities of the different clones werequantitatively assessed. Fluorescence was measured fluorimetrically forcells grown in liquid culture in the presence or absence of unnaturalamino acid (FIG. 23 b). The Cm IC₅₀s were determined by plating thecells on varying concentrations of Cm in the presence or absence ofunnatural amino acid (FIG. 23 c).

A myoglobin gene containing an amber codon in the fourth position wasused to assess the production of unnatural amino acid-containingprotein. The gene was expressed in cells, using the pIF-RS, pAF-RS, orOMY-RS variant, respectively, in either the presence or absence of pIF,pAF, or OAT (FIG. 23 d). Protein yields were comparable for all threevariants, ranging from 1-2 milligrams of protein per liter of unnaturalamino acid-containing cell culture. In contrast, protein production wasvirtually undetectable in cultures grown in the absence of unnaturalamino acid. Proteins were analyzed by electrospray mass spectrometry,giving masses of 18457.40±0.81 (18457.28 expected) for thepIF-containing protein, and 18430.30±0.27 (18430.21 expected) for thepAF-containing protein. Activity measurements obtained using the CmIC₅₀, fluorimetry, and protein expression analyses correlated well,however the activity of the pIF-RS appears to be somewhat underestimatedby fluorimetry. As compared to other assays, the disproportionately lowfluorimetry measurement for the pIF-RS variant, suggests that T7 RNApolymerase may be partially destabilized upon incorporation of the pIFanalogue, despite the apparent permissivity of the amber positionswithin the reporter (see, FIG. 17 c).

Utility of the multipurpose reporter system. The reporter systemdescribed here allows the use of a single multipurpose plasmid for bothpositive selection and negative screening, obviating the need to shuttleplasmids between alternating rounds of positive and negative selection.A total of only three rounds of positive selection and one round ofnegative screening were required to enable the identification ofsynthetase variants that selectively accept desired unnatural aminoacids. These features allow evolution experiments to be carried out in amatter of days. The screening system can be used to readily identifyactive synthetase variants using agar plates containing unnatural aminoacid and to individually assay the amino acid specificity of thevariants.

As described above, the T7 RNA polymerase/GFP system can be used toquantitatively compare the activities of synthetase variants. Theavailability of the three OAT-RS clones described here and a differentOAT-RS clone derived independently from the same library using apositive/negative selection based on CAT and barnase allows thepossibility of comparing the two different evolution systems in terms ofthe synthetase variants resulting from each. This analysis reveals thatthe three clones derived from positive selection and negative screeningexhibit slightly lower levels of fluorescence in the presence of OAT,but ˜10-fold lower background levels in the absence of the unnaturalamino acid. The fluorescence enhancement for cells grown in the presenceversus the absence of the unnatural amino acid is thus about 6-foldhigher for cells expressing OAT-RS(1) from selection and screening thanfor cells expressing the OAT-RS clone derived from positive/negativeselection using barnase. Although it is not clear whether this exampleis representative, these data suggest that the T7 RNA polymerase/GFPsystem may allow more stringency in selecting against synthetasevariants that are promiscuous towards natural amino acid substrates.However, the fluorescence enhancement for cells grown in the presenceversus the absence of an unnatural amino acid is expected to represent alower limit for the fidelity of unnatural amino acid incorporation, ascompetition of unnatural amino acids for being bound by an evolvedsynthetase variant would reduce binding of natural amino acids.Moreover, although high fidelity is clearly desirable, there is likelyto be a trade-off between fidelity and overall synthetase activity,which may depend on the desired application.

Generality of aminoacyl tRNA synthetase evolution. Previous results andthose presented here demonstrate that the amino acid side chain bindingpocket of the M. jannaschii TyrRS is quite malleable. The enzyme can beevolved to accommodate a variety of functionalities in place of thephenol side chain of tyrosine and can do so with high selectivity. Inthis application it was demonstrated that enzyme can be evolved toaccommodate an amine, isopropyl, or allyl ether functionality at thepara position of the tyrosine ring, instead of hydroxyl. It was notpossible to identify an enzyme variant that could accept the pCFunnatural amino acid. A second attempt to evolve a synthetase to acceptthe pCF amino acid was also unsuccessful. Using LC/MS analysis, pCFcould not be detected upon toluenization of E. coli cells grown in thepresence of the unnatural amino acid, suggesting that pCF is nottransported into cells or that it is metabolized upon entry.

Of the three successful evolution experiments described here, only theevolution of the OAT-RS resulted in the identification of more than oneactive clone. The OAT-RS evolution was also the experiment that producedthe most active synthetase variant. These results suggest that someamino acid specificities may be easier to select for than others. Thiscould be due, in part, to the relative difficulty of selectivelyrecognizing different unnatural amino acids in the context of the 20natural amino acids. It may be, for example, that pAF, due to itsstructural and electronic similarities to tyrosine, is more difficult toselectively recognize than OAT. This would explain why a greater numberof OAT-RS clones were identified than pAF-RS clones and why the pAF-RSclone is less active than the best OAT-RS clone.

Plasmid Construction. Plasmid pREP (FIG. 17 a) was constructed byinsertion of a BamHI/ApaLI overlap PCR fragment containing the T7 RNApolymerase gene upstream of an rrnB transcription termination region,followed by an ApaLI/AhdI overlap PCR fragment containing the araC geneand ara promoter region from the pBAD/Myc-His A plasmid (Invitrogen; fortranscriptional control of the T7 RNA polymerase gene) and the GFPuvgene (Clontech; upstream of the T7 terminator region and downstream ofthe T7 promoter) between the AhdI/BamHI sites of plasmid pACYC177 (NewEngland Biolabs). Plasmids pREP(1-12) were constructed by replacement ofan HpaI/ApaLI fragment of T7 RNA polymerase with overlap PCR fragmentscontaining amber mutations at the positions described. PlasmidpREP/YC-JYCUA was constructed by ligation of an AfeI/SacII fragment frompREP(10) and an EarI(blunted)/SacII fragment from pYC-J17 (Wang, L,Brock, A., Herberich, B. & Schultz, P. G., Science, 2001, 292, 498-500).The desired construct was identified following transformation into cellscontaining plasmid pQ screening for fluorescence.

Plasmid pQ was constructed by triple ligation of a AatII/SalI overlapPCR fragment containing the ScQRS downstream of the lac promoter regionand upstream of the E. coli QRS termination region, a SalI/AvaI overlapPCR fragment containing the S. cerevisiae tRNA(CUA)^(Gln) downstream ofthe lpp promoter region and upstream of an rrnC termination region, andthe AvaI/AatII fragment of pBR322 (New England Biolabs). Plasmid pQD wasconstructed by replacement of pQ fragment between BamHI and BglII with aBamHI/BglII fragment of the ScQRS (D291A) mutant.

Plasmid pBAD/JYAMB-4TAG was constructed by insertion of a PCR fragmentof the S4Amber mutant of myoglobin, containing a C-terminal 6His-tag,into the pBAD/YC-JYCUA plasmid, a hybrid of plasmid pYC-J17 (Wang, L,Brock, A., Herberich, B. & Schultz, P. G., Science, 2001, 292, 498-500)and pBAD/Myc-His A (Invitrogen) containing the gene for MjYtRNA_(CUA),and the pBAD promoter and cloning regions for heterologous expression ofan inserted gene.

Fluorimetric and cytometric analyses. Single colonies containing desiredplasmids were used to inoculate 2-mL GMML cultures containing theappropriate antibiotics, 0.002% Arabinose, and an appropriate unnaturalamino acid, if desired. Cultures were grown to saturation and cells (200mL) were pelleted and resuspended in 1 mL phosphate-buffered saline(PBS). Cell concentrations were analyzed by absorbance at 600 nm andfluorescence levels were measured at 505 nm with excitation at 396 nmusing a FluoroMax-2 fluorimeter. Cells suspended in PBS were analyzedcytometrically. To evaluate the permissivity of the amber positionswithin the T7 polymerase gene of pREP(10), the reporter plasmid wastransformed into a panel of suppressor strains, which were subsequentlyanalyzed fluorimetrically.

Evolution of aminoacyl-tRNA synthetase variants. M. jannaschii TyrRSvariants randomized at positions Y32, E107, D158, 1159, and L162 (Wang,L, Brock, A., Herberich, B. & Schultz, P. G., Science, 2001, 292,498-500) were transformed into DH10B E. coli cells (Life Technologies)containing pREP/YC-JYCUA to generate a library with a diversity of ˜10⁹.Transformants were allowed to recover in SOC medium for 60 min at 37°C., and were grown to saturation in LB medium. To begin an initialpositive selection, 2 mL of library culture, pelleted and resuspended inGMML medium, was used to inoculate 500 mL of GMML containing 25 ng/mLTetracycline (Tet), 35 ng/mL Kanamycin (Kn), and 1 mM pIF, pAF, pCF, orOAY. After incubation for 3 hours at 37° C., Cm was added to a finalconcentration of 75 μg/mL and cells were grown to saturation (˜48hours). For the second positive selection, a 100-mL GMML culturecontaining Tet, Kn, 75 μg/mL Cm, and 1 mM pIF, pAF, pCF, or OAY wasinoculated with cells from the initial positive selection (500 μL) andgrown to saturation at 37° C. (˜24-36 hours). In preparation fornegative screening, a 25-mL GMML culture containing Tet, Kn, and 0.02%arabinose (Ara) was inoculated with cells from the second positiveselection (100 μL, pelleted and resuspended in GMML) and grown tosaturation at 37° C. (˜24 hours). Ara-induced cells grown in the absenceof unnatural amino acids (1 mL) were pelleted and resuspended in 3 mL ofphosphate-buffered saline (PBS). Cells were sorted for lack ofexpression of GFPuv using a BDIS FACVantage TSO cell sorter with aCoherent Enterprise II ion laser with excitation at 351 nm and emissionsdetected using a 575/25 nm bandpass filter. Collected cells were dilutedin at least 10 volumes of LB, containing Tet and Kn, and grown tosaturation. To begin the third round of positive selection, 100 μL ofcells from the negative screen were pelleted, resuspended in GMML, andused to inoculate 25 mL of GMML containing Tet, Kn, and 1 mM pIF, pAF,pCF, or OAY. After incubation for 3 hours at 37° C., Cm was added to afinal concentration of 75 μg/mL and cells were grown to saturation (˜24hours). Following the third positive selection, cells were plated onGMML/agar containing Tet, Kn, 0.002% Ara, 0, 75, or 100 μg/mL Cm, and 0or 1 mM pIF, pAF, pCF, or OAY, and grown for 48 hours at 37° C.Expression and characterization of unnatural amino acid-containingproteins. DH10B cells co-transformed with pBAD/JYAMB-4TAG and theappropriate pBK plasmid were used to inoculate a 100-mL GMML starterculture containing Kn and Tet, which was grown to saturation. A 500-mLculture containing Kn, Tet, 0.002% Ara, 5 μM FeCl₃, and the desiredunnatural amino acid (or none) was inoculated with 50 mL of the starterculture and grown to saturation (˜18 hours). Cultures were pelleted,sonicated, and the myoglobin protein isolated according to the protocolof the QiaExpressionist (Qiagen) His-tag purification kit. Proteins wereanalyzed electrophoretically on a 12-20% gradient SDS polyacrylamide geland by electrospray mass spectrometry.

Example 9 Orthogonal tRNA/Threonyl-tRNA Synthetase Pair

This example illustrates the generation of an orthogonaltRNA/Threonyl-tRNA synthetase pair. FIG. 27 illustrates a threonyl-tRNAsynthetase from Therms thermophilus. This synthetase has two N-terminalediting domains, a catalytic domain and a C-terminal anticodon bindingdomain (659 amino acids). To generate the orthogonal synthetase based onthe T. thermophilus synthetase, the editing domain(s), N1 or N1 and N2was deleted from the synthetase to generate an N-truncated T.thermophilus ThrRS (475 amino acids). This synthetase has the samecatalytic activity but lacks the proofreading activity. The N-truncatedsynthetase was screened for activity. The N-truncated synthetase did notaminoacylate Escherichia coli tRNA.

Because, the T thermophilus tRNAThr was found to be a substrate forEscherichia coli Threonyl-tRNA synthetase, the T thermophilus tRNAThrwas mutated in order to generate an orthogonal pair. FIG. 28 illustratesthe mutations made in the tRNA. Specifically, C2G71 was mutated toA2U71. In vitro charging experiments demonstrate that this mutant is nota substrate for the E. coli Threonyl-tRNA synthetase but is a goodsubstrate for the T. thermophilus Threonyl-tRNA synthetase. Anothermutant was also constructed, which included the following mutations:C2G714A2U71 and G34G35U364C34G35U36 in order to generate an ambersuppressor tRNA. Other mutant tRNAs with modified anticodon loops inaddition to C2G71->A2U71 were also generated to suppress three and fourbase codons such as TGA, ACCA, ACAA, AGGA, CCCT, TAGA, and CTAG. Allthese tRNAs were not as good as substrate as the wild type tRNAThr (withA2U71) but can be improved by mutating the anticodon binding site of theT. thermophilus Threonyl-tRNA synthetase.

Example 10 Sequences of Exemplary O-tRNAs and O-RSs

Exemplary O-tRNAs comprise a nucleic acid comprising a polynucleotidesequence selected from the group consisting of: SEQ ID NO:1-3 and/or acomplementary polynucleotide sequence thereof. See, Table 5, Appendix 1.Similarly, example O-RS include polypeptides selected from the groupconsisting of: a polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 35-66 and a polypeptide encodedby a nucleic acid comprising a polynucleotide sequence selected from thegroup consisting of: SEQ ID NO:4-34 and a complementary polynucleotidesequence thereof.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

APPENDIX 1 TABLE 5 SEQ tRNA ID or # Sequences Notes RS 1CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCATGGCGCTGGTTC M. jannaschiitRNA AAATCCGGCCCGCCGGACCA mtRNA_(CUA) ^(Tyr) 2CCCAGGGTAG CCAAGCTCGG CCAACGGCGA CGGACTCTAA ATCCGTTCTCHLAD03; an optimized tRNA GTAGGAGTTC GAGGGTTCGA ATCCCTTCCC TGGGACCAamber supressor tRNA 3GCGAGGGTAG CCAAGCTCGG CCAACGGCGA CGGACTTCCT AATCCGTTCTHL325A; an optimized tRNA CGTAGGAGTT CGAGGGTTCG AATCCCTCCC CTCGCACCAAGGA framshift supressor tRNA 4ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGmutant TyrRS (LWJ16) RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCAGATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTACTTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGCAATTCATTATCCTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGGAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAGTAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 5ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG p-iPr-PheRS RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGGGATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATGTGCTTATGGAAGTCCTTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGTTATCATTATCTTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 6ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG p-NH₂-PheRS(1)RS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCAGATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTCCTTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTGTTCTCATTATTATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 7ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG p-NH₂-PheRS(2)RS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTACTATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTACGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCCGTTGCATTATGCTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 8ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGp-NH₂-PheRS(3a) RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCATATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCGGCCGCATTATCCTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 9ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGp-NH₂-PheRS(3b) RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTTATATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTCCTTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCAGAGTCATTATGATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 10ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGO-Allyl-TyrRS(1) RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTTCGATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTACGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATACGTATCATTATGCTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 11ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGO-Allyl-TyrRS(3) RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCCTATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTATGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATAATACGCATTATGGGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 12ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGO-Allyl-TyrRS(4) RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTACGATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTCATTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCAGACTCATTATGAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 13ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG p-Br-PheRS RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCATATAGGTTTTGAACCAAGT GGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTAAGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCCGTGTCATTATCATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 14ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG p-Az-PheRS(1)RS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGCTATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTCGGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGTGATTCATTATGATGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 15ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG p-Az-PheRS(3)RS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGGGATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTACTTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATACGTATTATTATGCTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 16ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG p-Az-PheRS(5)RS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTGATAGGTTTTGAACCAAGTGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTCCGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCAGATTCATTCTAGTGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 17ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetases to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGACATAGGTTTTGAACCAAGTincorporate m-acylGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAproteins (Ketone 3-4)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCA ATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGAATGCATTATCAAGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTITAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 18ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTTACATAGGTTTTGAACCAAGTincorporate m-acylGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAproteins (Ketone 3-7)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTCTATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGATATTCATTATACAGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 19ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTAATAGGTTTTGAACCAAGTincorporate m-acylGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGACAGATTTAAACGCCTATTTAAACCAGAAAproteins (Ketone 4-1)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGATATTCATTATTTAGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 20ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTAATAGGTTTTGAACCAAGTincorporate m-acylGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGACAGATTTAAAAGCCTATTTAAACCAGAAAproteins (Ketone 5-4)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGTCAGTTAATGTAATTCATTATTTAGGCGTTGATGTTGTAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 21ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTAATAGGTTTTGAACCAAGTincorporate m-acylGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGCCAGATTTATCAGCCTATTTAAACCAGAAAproteins (Ketone 6-8)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGATATTCATTATTTAGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 22ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTACAATAGGTTTTGAACCAAGTincorporate m-methoxyGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAproteins (OMe 1-6)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGATATTCATTATGCAGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 23ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTACAATAGGTTTTGAACCAAGTincorporate m-methoxyGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGTCCGATTTACCAGCCTATTTAAACCAGAAAproteins (OMe 1-8)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGATATTCATTATTTAGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 24ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTACAATAGGTTTTGAACCAAGTincorporate m-methoxyGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAproteins (OMe 2-7)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCA ATGGGGTTAAAGGCAAAATATGTTTATGGAAGTATGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTCATCACATTATGACGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 25ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCAAATAGGTTTTGAACCAAGTincorporate m-methoxyGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGCCAGATTTACACGCCTATTTAAACCAGAAAproteins (OMe 4-1)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCA ATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGAATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGATATTCATTATTTAGGCGTTGATGTTGACGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 26ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCACATAGGTTTTGAACCAAGTincorporate m-methoxyGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATphenylalanine intoGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAproteins (OMe 4-8)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGCATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATGGACACCATTATATAGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 27ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAGMutant synthetase to RSTTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTTACATAGGTTTTGAACCAAGTincorporate p-O-allylGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATtyrosine into proteinsGCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAA (Allyl)GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCA ATGGGGTTAAAGGCAAAATATGTTTATGGAAGTGCATTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGCAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATTGCGCACATTATTTAGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAG AGATTATAA 28ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG Aminoacyl tRNARS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTGGTATAGGTTTTGAACCAAGTsynthetase for theGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATincorporation of p-GCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAA benzoyl-L-GGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCAphenylalanine (p-ATGGGGTTAAAGGCAAAATATGTTTATGGAAGTTCCTTCCAGCTTGATAAGGATTAT BpaRS(H6))ACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATACGAGTCATTATCTGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 29ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG Aminoacyl tRNARS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTACGATAGGTTTTGAACCAAGTsynthetase for theGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATincorporation of p-GCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAazido-phenylalanineGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCA(p-Az-PheRS(3))ATGGGGTTAAAGGCAAAATATGTTTATGGAAGTAATTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCCGCTTCATTATCAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 30ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG Aminoacyl tRNARS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTACGATAGGTTTTGAACCAAGTsynthetase for theGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATincorporation of p-GCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAazido-phenylalanineGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCA(p-Az-PheRS(6))ATGGGGTTAAAGGCAAAATATGTTTATGGAAGTCTGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCCTCTTCATTATGAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 31ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG Aminoacyl tRNARS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTCTTATAGGTTTTGAACCAAGTsynthetase for theGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATincorporation of p-GCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAazido-phenylalanineGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCA(p-Az-PheRS(20)ATGGGGTTAAAGGCAAAATATGTTTATGGAAGTACTTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCCGGTTCATTATCAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 32ATGGACGAATTTGAAATGATAAAGAGAAACACATCTGAAATTATCAGCGAGGAAGAG Aminoacyl tRNARS TTAAGAGAGGTTTTAAAAAAAGATGAAAAATCTGCTACTATAGGTTTTGAACCAAGTsynthetase for theGGTAAAATACATTTAGGGCATTATCTCCAAATAAAAAAGATGATTGATTTACAAAATincorporation of p-GCTGGATTTGATATAATTATATTGTTGGCTGATTTACACGCCTATTTAAACCAGAAAazido-phenylalanineGGAGAGTTGGATGAGATTAGAAAAATAGGAGATTATAACAAAAAAGTTTTTGAAGCA(p-Az-PheRS(24))ATGGGGTTAAAGGCAAAATATGTTTATGGAAGTTCGTTCCAGCTTGATAAGGATTATACACTGAATGTCTATAGATTGGCTTTAAAAACTACCTTAAAAAGAGCAAGAAGGAGTATGGAACTTATAGAAGAGAGGATGAAAATCCAAAGGTTGCTGAAGTTATCTATCCAATAATGCAGGTTAATCCACTGCATTATCAGGGCGTTGATGTTGCAGTTGGAGGGATGGAGCAGAGAAAAATACACATGTTAGCAAGGGAGCTTTTACCAAAAAAGGTTGTTTGTATTCACAACCCTGTCTTAACGGGTTTGGATGGAGAAGGAAAGATGAGTTCTTCAAAAGGGAATTTTATAGCTGTTGATGACTCTCCAGAAGAGATTAGGGCTAAGATAAAGAAAGCATACTGCCCAGCTGGAGTTGTTGAAGGAAATCCAATAATGGAGATAGCTAAATACTTCCTTGAATATCCTTTAACCATAAAAAGGCCAGAAAAATTTGGTGGAGATTTGACAGTTAATAGCTATGAGGAGTTAGAGAGTTTATTTAAAAATAAGGAATTGCATCCAATGGATTTAAAAAATGCTGTAGCTGAAGAACTTATAAAGATTTTAGAGCCAATTAGAAAGA GATTA 33ATGAGCGATT TCAGGATAAT TGAGGAGAAG TGGCAGAAGG CGTGGGAGAAArchaeoglobus fulgidus RSGGACAGAATT TTTGAGTCCG ATCCTAATGA GAAGGAGAAG TTTTTTCTCAleucyl tRNA-synthetaseCAATTCCCTA TCCTTACCTT AATGGAAATC TTCACGCAGG TCACACGAGA (AFLRS)ACCTTCACAA TTGGCGATGC CTTCGCCAGA TACATGAGAA TGAAGGGCTACAACGTTCTC TTTCCCCTCG GCTTTCATGT TACGGGCACC CCAATCATTGGCCTTGCGGA GCTCATAGCC AAGAGGGACG AGAGGACGAT AGAGGTTTACACCAAATACC ATGACGTTCC GCTGGAGGAC TTGCTTCAGC TCACAACTCCAGAGAAAATC GTTGAGTACT TCTCAAGGGA GGCGCTGCAG GCTTTGAAGAGCATAGGCTA CTCCATTGAC TGGAGGAGGG TTTTCACCAC AACCGATGAAGAGTATCAGA GATTCATCGA GTGGCAGTAC TGGAAGCTCA AGGAGCTTGGCCTGATTGTG AAGGGCACCC ACCCCGTCAG ATACTGCCCC CACGACCAGAATCCTGTTGA AGACCACGAC CTTCTCGCTG GGGAGGAGGC AACTATTGTTGAATTTACCG TTATAAAGTT CAGGCTTGAA GATGGAGACC TCATTTTCCCCTGTGCAACT CTCCGTCCCG AAACCGTGTT TGGCGTCACG AACATCTGGGTAAAGCCGAC AACCTACGTA ATTGCCGAGG TGGATGGGGA AAAGTGGTTTGTGAGCAAAG AGGCTTACGA GAAGCTCACC TACACGGAGA AAAAAGTCAGGCTGCTGGAG GAGGTTGATG CGTCGCAGTT CTTCGGCAAG TACGTCATAGTCCCGCTGGT AAACAGAAAA GTGCCAATTC TGCCTGCAGA GTTTGTTGACACCGACAACG CAACAGGAGT TGTGATGAGC GTTCCCGCAC ACGCTCCTTTTGACCTGGCT GCCATTGAGG ACTTGAAGAG AGACGAGGAA ACGCTGGCGAAGTACGGAAT TGACAAAAGC GTTGTAGAGA GCATAAAGCC AATAGTTCTGATTAAGACGG ACATTGAAGG TGTTCCTGCT GAGAAGCTAA TAAGAGAGCTTGGAGTGAAG AGCCAGAAGG ACAAGGAGCT GCTGGATAAG GCAACCAAGACCCTCTACAA GAAGGAGTAC CACACGGGAA TCATGCTGGA CAACACGATGAACTATGCTG GAATGAAAGT TTCTGAGGCG AAGGAGAGAG TTCATGAGGATTTGGTTAAG CTTGGCTTGG GGGATGTTTT CTACGAGTTC AGCGAGAAGCCCGTAATCTG CAGGTGCGGA ACGAAGTGCG TTGTTAAGGT TGTTAGGGACCAGTGGTTCC TGAACTACTC CAACAGAGAG TGGAAGGAGA AGGTTCTGAATCACCTTGAA AAGATGCGAA TCATCCCCGA CTACTACAAG GAGGAGTTCAGGAACAAGAT TGAGTGGCTC AGGGACAAGG CTTGTGCCAG AAGGAAGGGGCTTGGAACGA GAATTCCGTG GGATAAGGAG TGGCTCATCG AGAGCCTTTCAGACTCAACA ATCTACATGG CCTACTACAT CCTTGCCAAG TACATCAACGCAGGATTGCT CAAGGCCGAG AACATGACTC CCGAGTTCCT CGACTACGTGCTGCTGGGCA AAGGTGAGGT TGGGAAAGTT GCGGAAGCTT CAAAACTCAGCGTGGAGTTA ATCCAGCAGA TCAGGGACGA CTTCGAGTAC TGGTATCCCGTTGACCTAAG AAGCAGTGGC AAGGACTTGG TTGCAAACCA CCTGCTCTTCTACCTCTTCC ACCACGTCGC CATTTTCCCG CCAGATAAGT GGCCGAGGGCAATTGCCGTA AACGGATACG TCAGCCTTGA GGGCAAGAAG ATGAGCAAGAGCAAAGGGCC CTTGCTAACG ATGAAGAGGG CGGTGCAGCA GTATGGTGCGGATGTGACGA GGCTCTACAT CCTCCACGCT GCAGAGTACG ACAGCGATGCGGACTGGAAG AGCAGAGAGG TTGAAGGGCT TGCAAACCAC CTCAGGAGGTTCTACAACCT CGTGAAGGAG AACTACCTGA AAGAGGTGGG AGAGCTAACAACCCTCGACC GCTGGCTTGT GAGCAGGATG CAGAGGGCAA TAAAGGAAGTGAGGGAGGCT ATGGACAACC TGCAGACGAG GAGGGCCGTG AATGCCGCCTTCTTCGAGCT CATGAACGAC GTGAGATGGT ATCTGAGGAG AGGAGGTGAGAACCTCGCTA TAATACTGGA CGACTGGATC AAGCTCCTCG CCCCCTTTGCTCCGCACATT TGCGAGGAGC TGTGGCACTT GAAGCATGAC AGCTACGTCAGCCTCGAAAG CTACCCAGAA TACGACGAAA CCAGGGTTGA CGAGGAGGCGGAGAGAATTG AGGAATACCT CCGAAACCTT GTTGAGGACA TTCAGGAAATCAAGAAGTTT GTTAGCGATG CGAAGGAGGT TTACATTGCT CCCGCCGAAGACTGGAAGGT TAAGGCAGCA AAGGTCGTTG CTGAAAGCGG GGATGTTGGGGAGGCGATGA AGCAGCTTAT GCAGGACGAG GAGCTTAGGA AGCTCGGCAAAGAAGTGTCA AATTTCGTCA AGAAGATTTT CAAAGACAGA AAGAAGCTGATGCTAGTTAA GGAGTGGGAA GTTCTGCAGC AGAACCTGAA ATTTATTGAGAATGAGACCG GACTGAAGGT TATTCTTGAT ACTCAGAGAG TTCCTGAGGAGAAGAGGAGG CAGGCAGTTC CGGGCAAGCC CGCGATTTAT GTTGCTTAA 34GTGGATATTG AAAGAAAATG GCGTGATAGA TGGAGAGATG CTGGCATATT MethanobacteriumRS TCAGGCTGAC CCTGATGACA GAGAAAAGAT ATTCCTCACA GTCGCTTACCthermoautotrophicumCCTACCCCAG TGGTGCGATG CACATAGGAC ACGGGAGGAC CTACACTGTCleucyl tRNA-synthetaseCCTGATGTCT ATGCACGGTT CAAGAGGATG CAGGGCTACA ACGTCCTGTT (MtLRS)TCCCATGGCC TGGCATGTCA CAGGGGCCCC TGTCATAGGG ATAGCGCGGAGGATTCAGAG GAAGGATCCC TGGACCCTCA AAATCTACAG GGAGGTCCACAGGGTCCCCG AGGATGAGCT TGAACGTTTC AGTGACCCTG AGTACATAGTTGAATACTTC AGCAGGGAAT ACCGGTCTGT TATGGAGGAT ATGGGCTACTCCATCGACTG GAGGCGTGAA TTCAAAACCA CGGATCCCAC CTACAGCAGGTTCATACAGT GGCAGATAAG GAAGCTGAGG GACCTTGGCC TCGTAAGGAAGGGCGCCCAT CCTGTTAAGT ACTGCCCTGA ATGTGAAAAC CCTGTGGGTGACCATGACCT CCTTGAGGGT GAGGGGGTTG CCATAAACCA GCTCACACTCCTCAAATTCA AACTTGGAGA CTCATACCTG GTCGCAGCCA CCTTCAGGCCCGAGACAATC TATGGGGCCA CCAACCTCTG GCTGAACCCT GATGAGGATTATGTGAGGGT TGAAACAGGT GGTGAGGAGT GGATAATAAG CAGGGCTGCCGTGGATAATC TTTCACACCA GAAACTGGAC CTCAAGGTTT CCGGTGACGTCAACCCCGGG GACCTGATAG GGATGTGCGT GGAGAATCCT GTGACGGGCCAGGAACACCC CATACTCCCG GCTTCCTTCG TTGACCCTGA ATATGCCACAGGTGTTGTGT TCTCTGTCCC TGCACATGCC CCTGCAGACT TCATAGCCCTTGAGGACCTC AGGACAGACC ATGAACTCCT TGAAAGGTAC GGTCTTGAGGATGTGGTTGC TGATATTGAG CCCGTGAATG TCATAGCAGT GGATGGCTACGGTGAGTTCC CGGCGGCCGA GGTTATAGAG AAATTTGGTG TCAGAAACCAGGAGGACCCC CGCCTTGAGG ATGCCACCGG GGAGCTATAC AAGATCGAGCATGCGAGGGG TGTTATGAGC AGCCACATCC CTGTCTATGG TGGTATGAAGGTCTCTGAGG CCCGTGAGGT CATCGCTGAT GAACTGAAGG ACCAGGGCCTTGCAGATGAG ATGTATGAAT TCGCTGAGCG ACCTGTTATA TGCCGCTGCGGTGGCAGGTG CGTTGTGAGG GTCATGGAGG ACCAGTGGTT CATGAAGTACTCTGATGACG CCTGGAAGGA CCTCGCCCAC AGGTGCCTCG ATGGCATGAAGATAATACCC GAGGAGGTCC GGGCCAACTT TGAATACTAC ATCGACTGGCTCAATGACTG GGCATGTTCA AGGAGGATAG GCCTTGGAAC AAGGCTGCCCTGGGATGAGA GGTGGATCAT CGAACCCCTC ACAGACTCAA CAATCTACATGGCATATTAC ACCATCGCAC ACCGCCTCAG GGAGATGGAT GCCGGGGAGATGGACGATGA GTTCTTTGAT GCCATATTCC TAGATGATTC AGGAACCTTTGAGGATCTCA GGGAGGAATT CCGGTACTGG TACCCCCTTG ACTGGAGGCTCTCTGCAAAG GACCTCATAG GCAATCACCT GACATTCCAT ATATTCCACCACTCAGCCAT ATTCCCTGAG TCAGGGTGGC CCCGGGGGGC TGTGGTCTTTGGTATGGGCC TTCTTGAGGG CAACAAGATG TCATCCTCCA AGGGCAACGTCATACTCCTG AGGGATGCCA TCGAGAAGCA CGGTGCAGAC GTGGTGCGGCTCTTCCTCAT GTCCTCAGCA GAGCCATGGC AGGACTTTGA CTGGAGGGAGAGTGAGGTCA TCGGGACCCG CAGGAGGATT GAATGGTTCA GGGAATTCGGAGAGAGGGTC TCAGGTATCC TGGATGGTAG GCCAGTCCTC AGTGAGGTTACTCCAGCTGA ACCTGAAAGC TTCATTGGAA GGTGGATGAT GGGTCAGCTGAACCAGAGGA TACGTGAAGC CACAAGGGCC CTTGAATCAT TCCAGACAAGAAAGGCAGTT CAGGAGGCAC TCTATCTCCT TAAAAAGGAT GTTGACCACTACCTTAAGCG TGTTGAGGGT AGAGTTGATG ATGAGGTTAA ATCTGTCCTTGCAAACGTTC TGCACGCCTG GATAAGGCTC ATGGCTCCAT TCATACCCTACACTGCTGAG GAGATGTGGG AGAGGTATGG TGGTGAGGGT TTTGTAGCAGAAGCTCCATG GCCTGACTTC TCAGATGATG CAGAGAGCAG GGATGTGCAGGTTGCAGAGG AGATGGTCCA GAATACCGTT AGAGACATTC AGGAAATCATGAAGATCCTT GGATCCACCC CGGAGAGGGT CCACATATAC ACCTCACCAAAATGGAAATG GGATGTGCTA AGGGTCGCAG CAGAGGTAGG AAAACTAGATATGGGCTCCA TAATGGGAAG GGTTTCAGCT GAGGGCATCC ATGATAACATGAAGGAGGTT GCTGAATTTG TAAGGAGGAT CATCAGGGAC CTTGGTAAATCAGAGGTTAC GGTGATAGAC GAGTACAGCG TACTCATGGA TGCATCTGATTACATTGAAT CAGAGGTTGG AGCCAGGGTT GTGATACACA GCAAACCAGACTATGACCCT GAAAACAAGG CTGTGAATGC CGTTCCCCTG AAGCCAGCCA TATACCTTGA ATGA35 MDEFEMIKRNTSEIISEEELREVLKKDEKSAQIGFEPSGKIHLGHYLQIKKMIDLQNmutant TyrRS(LWJ16) RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNAIHYPGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVSSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 36MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQN TyrRS (SS12)RS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPAHYQGVDVVVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTI 37MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDLQN p-iPr-PheRS RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKCAYGSPFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGYHYLGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 38MDEFEMIKRNTSEIISEEELREVLKKDEKSAQIGFEPSGKIHLGHYLQIKKMIDLQN p-NH₂-PheRS(1)RS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSPFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNCSHYYGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 39MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQN p-NH₂-PheRS(2)RS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPLHYAGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 40MDEFEMIKRNTSEIISEEELREVLKKDEKSAHIGFEPSGKIHLGHYLQIKKMIDLQNp-NH₂-PheRS(3a) RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVERPHYLGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 41MDEFEMIKRNTSEIISEEELREVLKKDEKSAQIGFEPSGKIHLGHYLQIKKMIDLQNp-NH₂-PheRS(3b) RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSPFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNQSHYDGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 42MDEFEMIKRNTSEIISEEELREVLKKDEKSASIGFEPSGKIHLGHYLQIKKMIDLQNO-Allyl-TyrRS(1) RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNTYHYAGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 43MDEFEMIKRNTSEIISEEELREVLKKDEKSAPIGFEPSGKIHLGHYLQIKKMIDLQNO-Allyl-TyrRS(3) RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSMFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNNTHYGGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 44MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQNO-Allyl-TyrRS(4) RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSHFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNQTHYEGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 45MDEFEMIKRNTSEIISEEELREVLKKDEKSAHIGFEPSGKIHLGHYLQIKKMIDLQN p-Br-PheRS RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSKFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPCHYHGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 46MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHLGHYLQIKKMIDLQN p-Az-PheRS(1)RS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSRFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNVYHYDGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 47MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDLQN p-Az-PheRS(3)RS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNTYYYLGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 48MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQN p-Az-PheRS(5)RS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSPFQLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNQIHSSGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL 49MDEFEMIKRNTSEIISEEELREVLKKDEKSADIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYincorporate m-acylTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGMHYQGVDVAVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins (Ketone 3-4)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 50MDEFEMIKRNTSEIISEEELREVLKKDEKSAYIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSLFQLDKDYincorporate m-acylTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNDIHYTGVDVAVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins (Ketone 3-7)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 51MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLTDLNAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYincorporate m-acylTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVAVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins (Ketone 4-1)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 52MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLTDLKAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYincorporate m-acylTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMSVNVIHYLGVDVVVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins (Ketone 5-4)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 53MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLPDLSAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYincorporate m-acylTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVAVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins (Ketone 6-8)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 54MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYincorporate m-methoxyTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNDIHYAGVDVAVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins (OMe 1-6)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 55MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLSDLPAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYincorporate m-methoxyTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVAVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins(OMe 1-8)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 56MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSMFQLDKDYincorporate m-methoxyTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNSSHYDGVDVAVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins (OMe 2-7)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 57MDEFEMIKRNTSEIISEEELREVLKKDEKSAQIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLPDLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEFQLDKDYincorporate m-methoxyTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNDIHYLGVDVDVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins (OMe 4-1)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 58MDEFEMIKRNTSEIISEEELREVLKKDEKSAHIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSAFQLDKDYincorporate m-methoxyTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGHHYIGVDVAVGGMphenylalanine intoEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKproteins (OMe 4-8)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 59MDEFEMIKRNTSEIISEEELREVLKKDEKSAYIGFEPSGKIHLGHYLQIKKMIDLQNMutant synthetase to RSAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSAFQLDKDYincorporate p-O-allylTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNCAHYLGVDVAVGGMtyrosine into proteinsEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKK (Allyl)AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKRL# 60MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDLQN Aminoacyl tRNARS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSFQLDKDYsynthetase for theTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNTSHYLGVDVAVGGMincorporation of p-EQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKK benzoyl-L-AYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM phenylalanineDLKNAVAEELIKILEPIRKRL (p-BpaRS(H6)) 61MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQN Aminoacyl tRNARS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSNFQLDKDYsynthetase for theTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPLHYQGVDVAVGGMincorporation of p-EQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKazido-phenylalanineAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM(p-Az-PheRS(3)) DLKNAVAEELIKILEPIRKRL 62MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQN Aminoacyl tRNARS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSFQLDKDYsynthetase for theTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPLHYQGVDVAVGGMincorporation of p-EQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKazido-phenylalanineAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM(p-Az-PheRS(6)) DLKNAVAEELIKILEPIRKRL 63MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHLGHYLQIKKMIDLQN Aminoacyl tRNARS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSTFQLDKDYsynthetase for theTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPVHYQGVDVAVGGMincorporation of p-EQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKazido-phenylalanineAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM(p-Az-PheRS(20)) DLKNAVAEELIKILEPIRKRL 64MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHLGHYLQIKKMIDLQN Aminoacyl tRNARS AGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSSFQLDKDYsynthetase for theTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNPSHYQGVDVAVGGMincorporation of p-EQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKazido-phenylalanineAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM(p-Az-PheRS(24)) DLKNAVAEELIKILEPIRKRL 65MSDFRIIEEK WQKAWEKDRI FESDPNEKEK FFLTIPYPYL NGNLHAGHTRArchaeoglobus fulgidus RSTFTIGDAFAR YMRMKGYNVL FPLGFHVTGT PIIGLAELIA KRDERTIEVYleucyl trna-synthetaseTKYHDVPLED LLQLTTPEKI VEYFSREALQ ALKSIGYSID WRRVFITTDE (AFLRS)EYQRFIEWQY WKLKELGLIV KGTHPVRYCP HDQNPVEDHD LLAGEEATIVEFTVIKFRLE DGDLIFPCAT LRPETVFGVT NIWVKPTTYV IAEVDGEKWFVSKEAYEKLT YTEKKVRLLE EVDASQFFGK YVIVPLVNRK VPILPAEFVDTDNATGVVMS VPAHAPFDLA AIEDLKRDEE TLAKYGIDKS VVESIKPIVLIKTDIEGVPA EKLIRELGVK SQKDKELLDK ATKTLYKKEY HTGIMLDNTMNYAGMKVSEA KERVHEDLVK LGLGDVFYEF SEKPVICRCG TKCVVKVVRDQWFLNYSNRE WKEKVLNHLE KMRIIPDYYK EEFRNKIEWL RDKACARRKGLGTRIPWDKE WLIESLSDST IYMAYYILAK YINAGLLKAE NMTPEFLDYVLLGKGEVGKV AEASKLSVEL IQQIRDDFEY WYPVDLRSSG KDLVANHLLFYLFHHVAIFP PDKWPRAIAV NGYVSLEGKK MSKSKGPLLT MKRAVQQYGADVTRLYILHA AEYDSDADWK SREVEGLANH LRRFYNLVKE NYLKEVGELTTLDRWLVSRM QRAIKEVREA MDNLQTRRAV NAAFFELMND VRWYLRRGGENLAIILDDWI KLLAPFAPHI CEELWHLKHD SYVSLESYPE YDETRVDEEAERIEEYLRNL VEDIQEIKKF VSDAKEVYIA PAEDWKVKAA KVVAESGDVGEAMKQLMQDE ELRKLGKEVS NFVKKIFKDR KKLMLVKEWE VLQQNLKFIENETGLKVILD TQRVPEEKRR QAVPGKPAIY VA* 66VDIERKWRDR WRDAGIFQAD PDDREKIFLT VAYPYPSGAM HIGHGRTYTV MethanobacteriumRS PDVYARFKRM QGYNVLFPMA WHVTGAPVIG IARRIQRKDP WTLKIYREVHthermoautotrophicumRVPEDELERF SDPEYIVEYF SREYRSVMED MGYSIDWRRE FKTTDPTYSRleucyl trna-synthetaseFIQWQIRKLR DLGLVRKGAH PVKYCPECEN PVGDHDLLEG EGVAINQLTL (MtLRS)LKFKLGDSYL VAATFRPETI YGATNLWLNP DEDYVRVETG GEEWIISRAAVDNLSHQKLD LKVSGDVNPG DLIGMCVENP VTGQEHPILP ASFVDPEYATGVVFSVPAHA PADFIALEDL RTDHELLERY GLEDVVADIE PVNVIAVDGYGEFPAAEVIE KFGVRNQEDP RLEDATGELY KIEHARGVMS SHIPVYGGMKVSEAREVIAD ELKDQGLADE MYEFAERPVI CRCGGRCVVR VMEDQWFMKYSDDAWKDLAH RCLDGMKIIP EEVRANFEYY IDWLNDWACS RRIGLGTRLPWDERWIIEPL TDSTIYMAYY TIAHRLREMD AGEMDDEFFD AIFLDDSGTFEDLREEFRYW YPLDWRLSAK DLIGNHLTFH IFHHSAIFPE SGWPRGAVVFGMGLLEGNKM SSSKGNVILL RDAIEKHGAD VVRLFLMSSA EPWQDFDWRESEVIGTRRRI EWFREFGERV SGILDGRPVL SEVTPAEPES FIGRWMMGQLNQRIREATRA LESFQTRKAV QEALYLLKKD VDHYLKRVEG RVDDEVKSVLANVLHAWIRL MAPFIPYTAE EMWERYGGEG FVAEAPWPDF SDDAESRDVQVAEEMVQNTV RDIQEIMKIL GSTPERVHIY TSPKWKWDVL RVAAEVGKLDMGSIMGRVSA EGIHDNMKEV AEFVRRIIRD LGKSEVTVID EYSVLMDASDYIESEVGARV VIHSKPDYDP ENKAVNAVPL KPAIYLE*

1.-114. (canceled)
 102. A method for identifying an orthogonal tRNA-tRNAsynthetase pair for use in an in vivo translation system of a secondorganism, the method comprising: introducing a marker gene, a tRNA andan aminoacyl-tRNA synthetase (RS) isolated or derived from a firstorganism into a first set of cells from the second organism; introducingthe marker gene and the tRNA into a duplicate cell set from the secondorganism; and selecting or screening for surviving cells or for cellsshowing a specific screening response in the first set that fail tosurvive or show said response in the duplicate cell set, wherein thefirst set and the duplicate cell set are grown in the presence of aselection or screening agent, wherein the surviving or screened cellscomprise the orthogonal tRNA-tRNA synthetase pair for use in the in thein vivo translation system of the second organism.
 103. The method ofclaim 102, wherein the comparing and selecting or screening comprises anin vivo complementation assay.
 104. The method of claim 102, whereinconcentration of the selection or screening agent is varied.
 105. Themethod of claim 102, wherein the first organism is a prokaryoticorganism.
 106. The method of claim 102, wherein the second organism is aprokaryotic organism.
 107. The method of claim 102, wherein the firstand second organism are different.
 108. The method of claim 102, whereinthe first organism is selected from the group consisting of:Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and aHalobacterium.
 109. The method of claim 102, wherein the second organismis Escherichia coli.
 110. The method of claim 102, wherein the selectingor screening comprises one or more positive or negative selection orscreening chosen from the groups consisting of: a change in amino acidpermeability, a change in translation efficiency, and a change intranslational fidelity, and wherein the one or more change is based upona mutation in one or more gene in an organism in which an orthogonaltRNA-tRNA synthetase pair are used to produce protein.
 111. The methodof claim 102, wherein the selecting or screening comprises selecting orscreening at least 2 selector codons within one or more selection geneor within one or more screening gene.
 112. The method of claim 111,wherein the at least 2 selector codons are in the same selection gene orthe same screening gene.
 113. The method of claim 111, wherein the atleast 2 selector codons are in different selection or screening genes.114. The method of claim 111, wherein the at least 2 selector codonscomprise different selector codons.
 115. The method of claim 111,wherein the at least 2 selector codons comprise the same selectorcodons.